ISC

ISC Biotechnology Previous Year Question Paper 2015 Solved for Class 12

Maximum Marks: 80
Time allowed: Three hours

• Candidates are allowed additional 15 minutes for only reading the paper. They must NOT start writing during this time.
• Answer Question 1 (Compulsory) from Part I and five questions from Part II, choosing two questions from Section A, two questions from Section B and one question from either Section A or Section B.
• The intended marks for questions or parts of questions are given in brackets [ ].
• Transactions should be recorded in the answer book.
• All calculations should be shown clearly.
• All working, including rough work, should be done on the same page as, and adjacent to the rest of the answer.

Part-1
(Answer all questions)

Question 1.
(a) Mention any one significant difference between each of the following : [5]
(i) Prokaryotic genome and Eukaryotic genome
(ii) Purine and Pyrimidine
(iii) Centrifugation and Crystallography
(iv) Glucose and Glycogen
(v) Codon and Cosmid

(b) Answer the following questions : [5]
(i) Why are Flavr Savor tomatoes preferred over natural tomatoes ?
(ii) State one gene – one enzyme hypothesis.
(iii) Why is the amino acid glycine said to be optically inactive ?
(iv) State Chargaff’s Law of DNA bases.
(v) What is electroporation ?

(c) Write the full form of each of the following: [5]
(i) PIR
(ii) SSB
(iii) STS
(iv) BAC
(v) GBB

(d) Explain briefly: [5]
(i) Insertional inactivation
(ii) Microprocessor
(iii) Supramolecular assembly
(iv) Callus .
(v) Cybrid
Answer:
(a) (i) Prokaryotic genome: In prokaryotic genome, a naked DNA is present, equal to single chromosome.

Eukaryotic genome: In eukaryotic genome, DNA is associated with histone proteins and the number of chromosome is 2 to numerous.

(ii) Purine: They are large size double ring structure e.g., Adenine, Guanine.

Pyrimidine: They are small in size, single ring structures e.g., Thymine, Cytosine.

(iii) Centrifugation: A process to separate small molecules by the action of centrifugal force, simply a physical phenomenon.

Crystallography: It is a technique of studying 3-D structure of macromolecules /atomic arrangement / crystal structure (proteins and nuclear acids) by placing in an intense beam of monochromatic X-rays, producing the regular pattern of reflections.

(iv) Glucose: It is a monosaccharide

Glycogen: It is a polymer formed by condensation of large number of glucose monomers.

(v) Codon: It is a sequence of three nitrogen bases in wRNA which determine the incorpo¬ration of a specific amino acid in a polypeptide chain

Cosmid: They are formed by integration of plasmid with bacterial ori, an antibiotic selection marker, and a cloning site with one or more ‘cos’ sites derived from (λ) lambda bacteriophage.

(b) (i) Flavr Savor tomatoes are genetically modified tomatoes characterised by delayed ripening and longer shelf life.

(ii) One gene-oneenzyme hypothesis is the idea that genes act to the production of enzymes with each gene responsible for producing a single enzyme that intum affects a single step in a metabolic pathway. Concept was proposed by George Beadle and Edward Lawric Tatum in 1941.

(iii) Glycine is optically inactive because it does not affect the plane of polarised light.

(iv) (i) Chargaff’s rule states that purines and pyrimidines are equal in amount ;.e.,A+G = C + T
(ii) Molar amount of purine adenine is always equal to molar amount of thymine. Similarly guanine is equal by cytosine
(iii) Sugar deoxyribosome and phosphate occur in equimolar proportion
(iv) The ratio of A + T/ G + Cis constant for a species.

(v) Electroporation: It is the technique of introducing DNA into the cell by a brief exposure to a very high voltage electric pulse.

(c) (i) PIR – Protein Information Resources

(ii) SSB – Single Strand Breaks

(iii) STS – Sequence Tagged Sites

(iv) BAC – Bacterial Artificial Chromosome

(v) GDB-Genomic Data Bank

(d) (i) Insertional inactivation: Harder problem to solve is to determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain self-ligated vector molecules. Insertional inactivation is the inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. Recombinants can therefore, be identified because the characteristic coded by the inactivated gene is no longer visible.

pBR322 contains genes which code for ampicillin resistance and tetracycline resistance. BamHI cuts in the middle of the gene which codes for tetracycline resistance. If a gene is inserted here, the plasmid loses its ability to code for tetracycline resistance. Thus, the plasmid containing the recombinant gene is resistant to ampicillin but sensitive to tetracycline. To screen, we use replica plates.

The pUC8 plasmid is ampicillin resistant and contains a gene lac Z’which partially codes for β galactosidase. To make the plasmid capable of coding for the whole protein, we add the missing DNA along with the recombinant gene. The host which contains the plasmid pUC8 is resistant to ampicillin and is also capable of producing β galactosidase.

(ii) Microprocessor: A microprocessor is a programmable digital electronic component that incorporates the functions of a central processing unit (CPU) on a single integrated circuit (IC).

(iii) Supra molecular assembly is based on weak bonds as supposed to covalent bonds. As mentioned weak bond, though harder to control have the advantage of possible reopening. Relying on weak bond in surface organisation, can lead to molecular assemblies and raise the probability of reorganization processes.

(iv) Callus: It is a mass of meristematic undifferentiated unorganized cells produced in a culture,

(v) Cybrids or a cytoplasmic hybrids are cells or plants containing nucleus of one species but cytoplasm from both the parental species.

Part — II
(Answer any five questions)

Question 2.
(a) Explain the general structure of mRNA and tRNA. Mention their functions during protein
synthesis. [4]
(b) Give one application of each of the following in genetic engineering techniques. [4]
(i) Restriction enzymes and DNA ligases
(ii) Shuttle vectors and Expression vectors
(c) What is meant by genetic code ? [2]
Answer:
(a) Formation of RNA over DNA template is called translation. It is meant for taking the coded information from DNA to the site in cytoplasm where it is required for protein synthesis. Only one of the two strands – sense strand transcribes it. mRNA brings instructions from the DNA for the formation of particular type of polypeptide. The instructions are present in the base sequence of nucleotide’s. Formation of polypeptide occurs over the ribosome, mRNA gets attached to the ribosome.

tRNA are induced to bring amino acids in a particular sequence according to the sequence of codons present over mRNA. mRNA has methylated region at 5′ terminus the cap for attachment with ribosomes. Then followed by initiation code (AUG) either immediately or after a small non-coding region. Then there is coding region followed by termination codon (UAA, UGA, UAG). Then there is non-coding region and poly A’ area at the 3′ terminus. mRNA may be monocistronic (Eukaryote) or polycistronic (Prokaryote).

Transfer RNA (tRNA) : It is also called soluble or sRNA. There are over 100 types of tRNAs. Transfer RNA constitutes about 15% of the total RNA. tRNA is the smallest RNA with 70-85 nucleotides and sedimentation coefficient of 4S. The nitrogen bases of several of its nucleotides get modified, causes coiling of the otherwise single- stranded tRNA into L-shaped form (three-dimensional, Klug, 1974) or clover-like form (two dimensional, Holley, 1965).

It has (i) Anti codon: It is made-up of three nitrogen bases for recognizing and attaching to the codon of mRNA.
(ii) AA-Binding Site : It lies at the 3′ end opposite the anti codon and has CCA—OH group (5′ end bears G). Amino acid or AA-binding site and anti codon are the two recognition sites of tRNA.

Functions : (i) tRNA is adapter molecule which is meant for transferring amino acids to ribosomes for synthesis of polypeptides. There are different tRNA’s for different amino acids. Codons are recognized by anti codons of fRNAs. Specific amino acids are recognized by particular activating or aminoacyl synthetase enzymes.
(ii) They hold peptidyl chains over the mRNAs. The initiator tRNA has the dual function of initiation of protein synthesis as well as bringing in of the first amino acids. There is, however, no tRNA for stop signals.

(b) (i) Restriction enzymes recognize and cleaves a specific short DNA sequence at recognition site. They are used in r-DNA technology as molecular scissors. DNA ligases are the enzymes used to join cut ends of vector DNA and the target DNA by their sticky ends.

(ii) Shuttle vectors is a plasmid containing vector, that can replicate in two different organisms- prokaryote and eukaryote, due to the presence of two different origin of replication i.e, Ori Euk, Ori E-coli.

Expression vectors in addition to incorporate desired gene into the host cell, also help to produce high amount of proteins enclosed by the DNA insert, because of presence of promoter sequence and a terminator sequence along with insert.

(c) Though made-up of only four types of nucleotides, nucleotides of DNA can be positioned in a countless way. As a result, single DNA molecule has several thousand nucleotides, a limitless specificity can be incorporated in the DNA.

There is intimate connection between genes and the synthesis of polypeptides. Genes are made- up of nucleotides, arranged in a specific manner. Arrangement of nucleotides or their nitrogen bases is connected with synthesis of proteins by influencing the corporation of amino acids in them. The relationship between the sequence of amino acids in a polypeptide chain and nucleotide sequence of DNA or mRNA is called genetic code. DNA contains only four types of nucleotide or nitrogen bases while the amino acid is 20. It was, therefore, hypothesized, the triplet code (consisting of three adjacent bases for one amino acid) is operative.

Question 3.
(a) With reference to lipids, explain the following: [4]
(i) Any two chemical properties of lipids.
(ii) Chemical structure and any one function of lipids.
(b) Give the causes and the symptoms of the following metabolic disorders : [4]
(i) Albinism
(ii) Sickle cell anaemia
(c) Name two low resolution techniques used in gene analysis. [2]
Answer:
(a) (i) Chemical properties of Lipids: Hydrolysis: On hydrolysis with alkali or lipolytic enzyme lipases, fats are broken down into their components fatty acids and glycerols.

Saponification Value : It is the amount in mg of alkali required to saponify 1 gm of fat or oil. This value is useful for a comparative study of fatty acid chain-length in oils.

(ii) Chemical structure of lipids: Lipids are a heterogeneous group of fats or fat like substances which are insoluble in water but soluble in non-polar solvents like ether, acetone, e.g., ghee, butter, oil, vitamins A, E and K.

Lipids are formed of carbon, hydrogen and often oxygen where oxygen content is very small as compared to other two elements. Lipids require more oxygen for their complete oxidation as compared to carbohydrates. Some lipids also contain small amount of phosphorus nitrogen and rarely sulfur. Most lipids are formed of fatty acids.

Lipids are water insoluble components of the living cells and are essential for storing surplus food. They provide insulation to heat loss and protect internal organs from abrasion. Most biological membranes contain around 50-40% of lipids. Some lipids act as carrier of some vitamins (vitamin A,D, E and K) and also protect them from oxidation. In animals, the fats produce a shock absorbing cushion around eye balls, gonads, kidneys and other vital organs.

(b) (i) Albinism : It is caused by the absence of the enzyme tyrosinase which is essential for the synthesis of the pigment from dihydroxyphenyalanine. The gene for albinism (a) does not produce the enzyme tyrosinase but its normal allele (A) does. Thus, only homozygous individual (aa) is affected by this disease. Albinos (individuals with albinism) lack dark pigment melanin in the skin, hair and iris. Although albinos have poor vision yet they lead normal life.

(ii) Sickle cell anaemia is an autosomal hereditary disorder due to a mutation of single nitrogen base. It results the formation of an abnormal haemoglobin called haemoglobin S (Hb^s). In this, only one amino acid-6th amino acid of β-chain glutamic acid is replaced by valine. The erythrocytes become sickle shaped under oxygen deficiency as during strenous exercise and at high altitudes. They cannot pass through narrow capillaries. They clog blood capillaries. Blood circulation and oxygen supply is disturbed. Spleen and brain get damaged. Patient feels acute weakness. Homozygotes (Hb^s / Hb^s) usually die before reaching maturity.

(c) (i) AFLP genotyping and fingerprinting
(ii) Enzyme electrophoresis
(iii) PCR – SSP technique

Question 4.
(a) With respect to tissue culture techniques, discuss the following : [4]
(i) Various sterilization techniques used.
(ii) Composition of culture medium.
(b ) Discuss the impact of the following factors on enzyme activity : [4]
(i) Substrate concentration and Enzyme concentration
(ii) Temperature and pH
(c) What is single nucleotide polymorphism 9 [2]
Answer:
(a) (i) Various Sterilization Technique :
Maintenance of Aseptic Environment: Dining in vitro culture maintenance of aseptic environment is the most difficult task, because the cultures are easily contaminated by fungi and bacteria present in the air. The contaminants produce toxic metabolites which inhibit growth of cultures plant tissues. Therefore, each step must be handled aseptically and with great care. Following are some of the sterilization methods for aseptic manipulation of plant tissues.

(a) Sterilization of glassware : Glassware (Petri plates, vials, culture tubes, flasks, pipettes, etc.), metallic instruments are sterilized in a hot air oven at 160-180° C for 2-4 hours.

(b) Sterilisation of instruments : The metallic instruments (e.g., forceps, scalpels, needles, spatulas, etc.) are flame sterilised i.e., dipping them in 25% ethanol followed by flaming and cooling. It is called incineration.

(c) Sterilisation of culture room and transfer area: Floor and walls of culture room are washed first with detergent then 2% sodium hypochlorite or 95% ethanol. Larger surface area is sterilised by exposure to UV light. The cabinet of laminar airflow is also sterilised by exposing UV light for 30 minutes and 95% ethanol 15 minutes before beginning of work inside the cabinet of laminar airflow.

(d) Sterilization of nutrient media : Culture media are properly dispensed in glass container, plugged with cotton or sealed with plastic closures and sterilized by autoclaving (steam sterilization) at 15 psi (that gives 121°C) for 30 minutes.

During autoclaving vitamins, plant extracts, amino acids and hormones are denatured. Therefore, the solution of these compounds are sterilized by using millipore filter paper which has 0.2 pm pore diameter.

(e) Sterilization of plant materials : Surface of all plant materials has microbial contaminants. Therefore, disinfectants (e.g., sodium hypochlorite, hydrogen peroxide, mercuric chloride, or ethanol) should be used to make plant materials sterile. Then the chemicals must be washed 6-8 times using sterile distilled water. Then ex plants are transferred aseptically on nutrient medium inside the cabinet of laminar airflow.

(ii) Composition of Nutrient Media: Composition of nutrient media governs the growth and morphogenesis of plant tissue in vitro. Depending on the type of plant cells or tissue used for culture the composition of nutrient media vary. The principal constituents of tissue culture media are inorganic nutrients, carbon sources, organic supplements, growth regulators and gelling agent.

(a) Inorganic nutrients: Several minerals (i.e., macro-and micro-nutrients) are required by the plants. Minerals dissolved in water are dissociated and ionized. For example, in MS medium NH₄N0₃ contributes NO^-3 and KNO₃ contributes K⁺ ions. There are six major macro-nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium and sulfur. But the essential or micro-nutrients are required in low amount. These are boron, molybdenum, cobalt, zinc, manganese, iron and copper.

(b) Carbon and energy sources: Mostly sucrose is required as carbon source followed by glucose. These carbon sources enhance cell proliferation and tissue regeneration.

(c) Organic supplements : There is a large number of organic supplements used for best growth of tissues. Vitamins (riboflavin, folic acid ) Amino acids (casein hydrolysate, L-glutamine, L-asperagine, L-glycine, L-arginine, L-cysteine) are commonly used as nitrogen source and enhancers of cell growth. Besides, culture media are also supplemented with casein, coconut milk, yeast and malt extracts, ground banana, orange juice and tomato juice. Activated charcoal added to culture media is known to stimulate plant growth. If required antibiotics (streptomycine or kanamycin) may be added to culture medium to avoid systemic infection of micro-organisms.

(d) Growth regulators : For proliferation of cultured tissues four classes of growth regulators (e.g., auxins, cytokinins, gibberellins and abscisic acid) are used. For induction of root or shoot the ratio of hormones varies considerably. For example, auxins (e.g., indole acetic acid, 1-naphthaleneacetic acid) induce cell division and cause elongation of stem, and intemodes. Cytokinins (e.g., 6-benzylaminopurine, 6-benzyladenine, zeatin) induce cell division and shoot differentiation of cultured tissues. Different ratios of auxins and cytokinins are important in morphogenesis of callus. High ratio of auxin to cytokinin promotes embiyogenesis, callus and root initiation. But high ratio of cytokinin to auxin leads to axillary and shoot promotion.

(e) Solidifying agents : Most commonly agar is used as solidifying or gelling agent. Agar gels do not react with constituents of media and not digested by plant enzymes. Generally 0.5 -1% agar is used to form gel.

(b) (i) Substrate concentration : It depends on the type of enzyme reaction. For a competitive enzyme reaction, the increase in substrate concentration can have an inverse effect on the activity, but for a normal enzyme reaction, as long as the & substrate concentration isn’t saturated, in general it will increase activity. At lower concentrations, the active sites on most of the enzyme molecules are not filled because there is not much substrate. Higher concentrations cause more-collisions between the molecules. With more molecules and collisions, enzymes are more likely to encounter molecules of reactant. The maximum velocity of a reaction is reached when the active sites are almost continuously filled. Increased substrate concentration after this point will not increase the rate. Reaction rate, therefore, increases as substrate concentration is increased but it levels off.

Enzyme Concentration : If there is insufficient enzyme present, the reaction will not proceed as fast as it otherwise would because all of the active sites are occupied with the reaction. Additional active sites could speed up the reaction. As the amount of enzyme is increased, the rate of reaction increases. This is because when more enzyme molecules are present,more substrate molecules can be acted upon at the same time. This means that the total substrate molecules are broken down quickly. If there are more enzyme molecules than are needed, adding additional enzyme will not increase the rate. Reaction rate therefore increases as enzyme concentration increases but then its levels off.

(iii) Temperature : Higher temperature generally causes more collisions among the molecules and therefore, increases the rate of a reaction. More collisions increase the likelihood that substrate will collide with the active site of the enzy me, thus increasing the rate of an enzyme-catalyzed reaction.

Above a certain temperature, activity begins to decline because the enzyme begins to denature. The rate of chemical reactions, therefore, increases with temperature but then decreases as enzymes denature.

Optimum pH : Every enzyme has an optimum pH when it is most effective. A rise or fall in pH reduces enzyme activity by changing the degree of ionization of its side chains. A change in pH may also start reverse reaction. Fumarase catalyses fiimarate → malate at 6.2 pH and reverse at 7.5 pH. Most of the intracellular enzymes function near neutral pH with the exception of several digestive enzymes which work either in acidic range of pH or alkaline, e.g., 2.0 pH for pepsin, 8.5 for trypsin.

(c) Single Nucleotide Polymorphisms (SNPs): SNPs are the variations in a nucleotide at genomic DNA in different individuals at a population which occur due to change even in a single base (e.g.. A, G, T or C). Therefore, certain sites of the sequence nucleotide bases of different
individuals differ as below :
First person — …ATGCTACG…
Second person — …TATCTACG…

In human genome, SNPs occur at 1.6-3.2 million sites. According to changes in bases SNPs affect the gene function. DNA fingerprinting of individuals is possible due to these genetic variations in non-coding parts of genome. This technique is used in search of criminals, rapists, solving parentage problem, confirming identity of individuals, etc.

On average, SNPs occur at every 500-1,000 nucleotides in human DNA, SNPs can help to

• associate sequence variation with heritable phenotypes,
• facilitate studies in population, and
• evolutionary biology, and add in positional cloning and physical mapping.

Question 5.
(a) List the functions of the following bioinformatics tools : [4]
(i) Taxonomy Browser
(ii) BLAST
(iii) ENTREZ
(iv) EMBL
(b) Explain the principle and applications of the following biochemical techniques : [4]
(i) Gel permeation
(ii) Electrophoresis
(c) Give two reasons for germplasm conservation. [2]
Answer:
(a) (i) Taxonomy browser: This search tool provides taxonomic information on various species. The TAXONOMY database of NCBI has information (including scientific and common names) about all organisms for which some sequence information is available (over 79,000 species). The server provides genetic information and the taxonomic relationship of the species in question. TAXONOMY has links with other servers of NCBI e.g., structure and PubMed.

(ii) BLAST (Basic Local Alignment Search Tool) : BLAST is a family of user-friendly sequence similarity search tools on the web. The BLAST server is supported through NCBI (National Center for Biotechnology Information) U.S.A. This tool is designed to identify potential homologous for a given sequence. It can analyse both DNA and protein sequences. A local alignment finds the optimal alignment between subregions or local regions of specified sequences.

(iii) ENTREZ: Integrated information database retrieval system of NCBI. Using Entrez system you can access literature, sequences (both proteins and nucleotides) and structures (3D).

(iv) EMBL (European molecular Biology Laboratory- Nucleic Acid (DNA sequence) databases.

(b) (i) Gel permeation or gel filtration : Size exclusion chromatography (SEC) is a cinematographic method in which particles are separated based on their size, or in more technical terms, their hydrodynamic volume. The name gel permeation chromatography is used when an organic solvent is used as a mobile phase. The main application of gel permeation chromatography is used to analyse the molecular weight distribution of organic-soluble polymer.

Theory and method : The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size, provided that all the particles are loaded simultaneously or near simultaneously.

Particles of the same size should elute together. This is usually achieved with an apparatus called a column which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the less overall volume to traverse over the length of the column and the faster the elution.

The filtered solution that is collected at the end is known as the elute. The void volume includes any particles too large to enter the medium and the solvent volume is known as the column volume.

(ii) Electrophoresis : It is the method of moving charged particles through a medium under the influence on an electric field. It is also used to separate molecules with different physical characteristics using electrical charges.

Types :

• Capillary electrophoresis — commonly used to separate biomolecules by their charge and frictional forces.
• Gel electrophoresis — a technique used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. It can be used as a preparative technique to partially purify- molecules prior to use of other methods such as mass spectrometry. PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

Examples of specific techniques include :

• DNA electrophoresis — a specific type of gel electrophoresis used to analyse DNA.
• Protein electrophoresis — a specific type of gel electrophoresis used to analyse proteins.
• Two-dimensional gel electrophoresis — a specific type of gel electrophoresis commonly used to analyse proteins which involves two separation mechanisms to separate molecules.
• SDS PAGE, (sodium dodecyl sulphate polyacrylamide gel electrophoresis) — commonly used to analyse proteins.

(c) Germplasm Conservation : The sum total of all the genes present in a crop and its related species constitutes its germplasm; it is ordinarily represented by a collection of various strains and species. Germplasm provides the raw materials (= genes), which the breeder uses to develop commercial crop varieties. Therefore, germplasm is the basic indispensable ingredient of all breeding programmes, and a great emphasis is placed on collection, evaluation and conservation of germplasm.

Question 6.
(a) Explain the principle and procedure of the PCR technique
(b) Discuss the following innovations in Biotechnology-:
(i) Oil eating bacteria
(ii) Recombinant insulin
(c) What is distant hybridization ?
Answer:
(a) Polymerase chain reaction (PCR) technique was originally invented by Kary Mullis (1985). It results in the selective amplification of a specific region of DNA molecule (up to billion copies). It can generate micro-gram (pg) quantities of DNA copies. The PCR process has been completely automated and compact thermal cycles are available in market.

Principle : The basic principle underlying this technique is that when a double strand DNA molecule is heated to a high temperature, the two DNA strands separate, giving rise to a single stranded DNA molecules (templates). If these single stranded molecules are copied by a DNA polymerase, it would lead to the duplication of original DNA molecule and if these events are repeated many times, multiple copies of the original DNA sequence can be regenerated.

The PCR is carried out in vitro. It utilises the following:

• a DNA preparation containing the desired segment to be amplified (target sequence),
• two nucleotide primers (about 20 bases long) specific, i.e., complementary, to the two 3′-borders (the sequences present at or beyond the 3′-ends of the two strands) of the desired segment,
• the four deoxynucleoside triphosphates, viz. TTP (thymidine triphosphate, dCTP (deoxycytidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate), and
• a heat stable DNA polymerase, e.g., Taq (isolated from the bacterium Thermus acquaticus), Pfu (from Pyrococcus furiosus) and Vent (from Thermococcus litoralis) polymerases. Pfu and Vent polymerases are more efficient than the Taq polymerase.

Procedure of PCR:
At the start of PCR, the DNA from which a segment is to be amplified, an excess of the two primer molecules, the four deoxyriboside triphosphates and the DNA polymerase are mixed together in the reaction mixture that has appropriate quantities of Mg²⁺. The following operations are now performed sequentially (Fig.)

Denaturation:
The reaction mixture is first heated to a temperature between 90-98°C (commonly 94°C) that ensures DNA denaturation. This is the denaturation step. The duration of this step in the first cycle of PCR is usually 2 min at 94°C.

Annealing:
The mixture is now cooled to a temperature (generally 40-60°C) that permits annealing of the primer to the complementary sequences in the DNA; these sequences are located at the 3′-ends of the two strands of the desired segment. This step is called annealing. The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. Since the primer concentration is kept very high relative to that of the template DNA, primer-template hybrid formation is greatly favored over reannealing of the template strands.

Primer Extension:
The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing 3′-OH of the primers; this reaction is the same as that occurs in vivo during replication of the leading strand of a DNA duplex. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3 -ends of the segment to be amplified. The duration of primer extension is usually 2 min. at 72°C. It has been shown that in case of longer target sequences, best results are obtained when the period of extension is kept at the rate of 1 min per kb of the target sequence and the extension is carried out at 68°C in the place of usual 72°C. Taq polymerase usually amplifies DNA segments of upto 2 kb; special reaction conditions are necessary’ for amplification of longer DNA segments.

In case of Taq polymerase, the optimum temperature for synthesis is between 70° and 75°C ; the temperature of reaction mixture is, therefore, adjusted to this temperature.

The completion of the extension step completes the first cycle of amplification; each cycle may take few’ (ordinarily 4-5) minutes. It should be noted that extension of the primer continues till the strands are separated during the denaturation step of the next PCR cycle.

The next cycle of amplification is initiated by denaturation (Step 1), which separates the newly synthesised DNA strands from the old DNA strands. Synthesis of new strands takes place, which doubles the number of copies of the desired DNA segment present at the end of first amplification cycle. This completes the second cycle.

Thus at each cycle, both new and old strands anneal to the primers and serve as templates for DNA synthesis. As a result, at the end of each cycle, the number of copies of the desired segment becomes twice the number present at the end of the previous cycle. Thus at the end of n cy cles 2” copies of the segment are expected; the real values are quite close to the expectation. Usually 20-30 cycles are carried out in most PCR experiments.

In case of automated PCR machines, called thermal cyclers, the researcher has to only specify the number and duration of cycles, etc. after placing the complete reaction mixture for incubation, and the machine performs the entire programme of operations precisely. After PCR cycles, the amplified DNA segment is purified by gel electrophoresis and can be used for the desired purpose.

PCR is of immense value in generating abundant amount of DNA for analysis in the DNA fingerprinting technique used in forensic science to link a suspect’s DNA to the DNA recovered at crime scene, to solve murder and rape cases etc.

Applications of Polymerase Chain Reaction Technology :

• PCR enables rapid amplification of template DNA for screening of uncharacterised mutation
• PCR permits rapid genotyping for polymorphic markers.
• A wide variety of PCR based methods can be used to assay for known mutation.
• Degenerate oligonucleotide primers and primers specific for ligated linker sequences permit co-amplification of sequence families.
• Anchored PCR uses a target specific primer and a universal primer for amplifying sequences adjacent to a known sequence.
• PCR is used in molecular diagnostics.

(b) An oil eating bacteria: Pseudomonas capacia, P. putida and other spp. Acinetobacter sp. Efficient degraders have been prepared through genetic engineering and they need to be established in the environment at a required density.

Production of Human Insulin (Humulin): Human insulin is a dimer comprising one chain of 21 amino acids (A chain) and the other of 30 amino acids (B chain), the C chain that links A and B chains. Chains A and B become linked by two disulphide bridges. This is followed by cleavage of the leader and the C chain sequences leaving the mature insulin molecule. Insulin is the first genetically engineered hormonal drug ever marketed anywhere in the world. It was produced first in 1980 by Eli Lilly (U S A.) with the name Humulin by transferring the insulin gene into E.coli.

The genes (= DNA sequences) for chains A and B of insulin were synthesized separately as early as 1978. The genes for A and B chains were integrated separetely in pBR322 type vector. The purified chains A and B were then attached to each other by disulphide bonds induced in vitro; this, however turned out to be an inefficient reaction. Subsequently, a gene representing B, C and A chains was synthesized and expressed in E. coli; in this case, the intervening chain is removed proteolytically following spontaneous folding of the proinsulin molecule.

(c) Distant hybridization : It implies crosses of species, subspecies, breeds and strains for the purpose of obtaining marketable hybrids of the first generation.

Importance of distant hybridization:
Distant hybridization is a combination of valuable features of parental forms in hybrids without noticeable increase in viability and, with intermediate growth rate.

They comprise both heterosis hybrids and hybrid forms with a favorable combination of parental features.

Abortion of embiyos at one or the other stage of development is a characteristic feature of distant hybridization, e.g., the crossing of two species or varieties often fails when using distant parents who do not share many chromosomes. Embryo rescue is the process when plant breeders rescue inherently weak, immature or hybrid embryos to prevent degeneration. Common in lily, hybridizing to create new inter specific hybrids between the various lily groups (such as Asiatic, Oriental, Trumpet, etc ).

Question 7.
(a) Given below is a list of four biomolecules. For each of them, write the class of biomolecules they belong to and their location in a living cell. [4]
(i) Cellulose
(ii) Histones
(iii) rRNA
(iv) Cholesterol
(b) What is freeze preservation ? Discuss any three types of freeze preservation. [4]
(c) Name any four important equipment used in cell culture technology. [2]
Answer:
(a)

(b) Freeze Preservation: In this approach, cells and tissues are stored at -196°C in liquid nitrogen. Theoretically, the cells and tissues can be stored in live state indefinitely, and cells/tissues recovered after thawing should be unchanged and live. But in practice, viability generally declines with the duration of cryopreservation, and thawed cells/tissues show variable degrees of damage, which they may be able to repair under appropriate media and culture conditions. It is desirable to preserve organised tissues like shoot-tips, and somatic and zygotic embryos to minimize the risk of genetic instability associated with unorganized tissues like cell suspensions and protoplasts.

(c) Equipment used in Cell Culture Technology :

• Washing and storage facilities.
• Media preparation room.
• Transfer area (Inoculation chamber / Laminar airflow, a closed plastic box).
• Culture room (Controlled system of light and temperature).

Note: Data collection: Cultures are monitored at regular intervals for growth and development of cultured tissue.

Question 8.
(a) Enumerate the process of DNA replication in living cells. Why is the DNA replication called semi-discontinuous ? [4]
(b) Explain how cell culture technology has been helpful in developing following traits in plants :
(i) Pest resistance
(ii) Drought resistance [4]
(c) What are the basic criteria in selecting an organism for its genome sequencing ? [2]
Answer:
(a) Mode of DNA Replication
DNA replication is a multi step complex process which requires over a dozen enzymes. It begins at a particular spot called origin of replication. Bacterial and viral DNA has a single origin of replication but in eukaryotic DNA there are a number of origin of replication because of the large size as well as association with proteins. Replication takes place as follows :

Activation of deoxyribonucleotides : Deoxyribonucleotides or deoxyribonucleoside monophosphates occur freely inside the nucleoplasm. They are first phosphorylated and changed to active forms which have three phosphate residues instead of one. Enzymes phosphorylase is required alongwith energy. The phosphorylated nucleotides are cfeATP. (deoxyadenosine triphosphate), deGTP (deoxyguanosine triphosphate), deCTP (deoxycytidine triphosphate) and deTTP (deoxythymidine triphosphate).

Exposure of DNA strands : Enzy mes topoisomerases are specialized to cause nicks (or breaks) and reseal the same in one strand of DNA. Helicases (unwindases) unwind the DNA helix and separate the two strands. In prokaryotes topoisomerases and helicases are replaced by DNA gyrases. The separated strands are stabilized by helix destabilizing (HD) or DNA binding proteins (DNP). They become open for replication. However, whole of DNA does not open in one stretch but the point of separation proceeds slowly from one end to the other. It gives the appearance of Y-shaped structure called replication fork.

RNA primer. It is essential for initiation of new DNA chains. RNA primer is a small strand of RNA which is synthesized at the 5′ end of new DNA strand with the help of enzyme called primase. It is specific. RNA primer is formed on the free end of one strand and fork end of the other strand. After start of nucleotide chain, RNA primer is removed and the gap is filled.

Base pairing: The two separated DNA strands in the replication fork function as templates. Deoxyribonucleoside triphosphates come to lie opposite the nitrogen bases of exposed DNA templates – deTPP opposite A, deCYP opposite G deATP opposite T and deGTP opposite C. With the help of enzyme pyrophosphatase the two extra phosphates present on the deoxyribonucleotides separate. Energy is released in the process. The energy is used in establishing hydrogen bonds between the free nucleotides and nitrogen bases of templates.

Chain formation : It requires DNA polymerase III (Komberg, 1956). In the presence of Mg²⁺, ATP (GTP), TPP and DNA polymerase III, the adjacent nucleotides present attached to nitrogen bases of each template DNA strand establish phosphodiester bonds and get linked to form replicated DNA strand. As replication proceeds new areas of parent DNA duplex unwind and separate so that replication proceeds rapidly from the place of origin towards the other end. RNA primer is removed that the gap filled with complementary nucleotides by means of DNA polymerase I. Because of sequential opening of DNA double chain and its replication to form two chains. DNA replication is also called Zipper duplication. However, DNA-polymerase can polymerise nucleotides only in 5′ direction because it adds them at the 3′ end. Since the two strands of DNA run in antiparallel directions, the two templates prov ide different ends for replication. Replication over the two templates thus proceeds in opposite directions. One strand is formed continuously because its 3′ end is open for elongation. It is called leading strand.

Replication is not continuous on the other template because only a short segment of DNA strand can be built in 5′ → 3′ direction due to exposure of a small stretch of template at one time. Short segments of replicated DNA are called Okazaki fragments. An RNA primer is also required every time a new Okazaki fragment is to be built. After replacing RNA primer the deoxv ribonucleotides and their polymerization. Okazaki fragments are joined together by means of enzyme. DNA ligase. DNA strand built-up of Okazaki fragments is called lagging strand.

Proof-reading and DNA repair: A wrong base is sometimes introduced during replication. The frequency is one in ten thousand. DNA polymerase is able to sense the same. It goes back, removes the wrong base, allows addition of proper base and then proceeds forward. However, even DNA polymerase III is unable to distinguish uracil from thymine so that it is often incorporated in place of thymine. Such a mismatching is corrected by means of a number of enzymes. DNA replication is semi-conservative. One half of the parent structure passes up to each replica while the second half is built a new.

(b) (i) Pest resistance : There is a large number of mites and insects that attack crop plants and cause great loss in quality and yield. To reduce the loss by the way of killing insects, farmers apply insecticides (i.e., synthetic pesticides) on the crop plants. However, the synthetic insecticides pose a serious threat to the health of plants, animals and humans. The alternative and novel ways of rescue from damages of insects are the use of transgenic technology. It is eco-friendly, cost-effective, sustainable and effective way of insect control. The cry genes of Bacillus thuringiensis (commonly called Bt gene) was found to express proteinaceous toxin inside the bacterial cells. When specific insects (species of Lepidoptera, Diptera, Coleoptera, etc.) ingest the toxin, they are killed. Toxin denatures the epithelium of gut by creating many holes at alkaline pH (7.5 to 8).

Using biotechnological approaches many transgenic crops having cry gene i.e., Bt-genes have been developed and commercialised. Some examples of Bi-crops are brinjal, cauliflower, cabbage, canola, com, cotton, egg plant, maize, potato, tobacco, tomato, rice, soyabean, etc.

(ii) Production of drought resistance : The level of water is gradually going down due to low rains and high temperature. Moreover, the plants requiring substantial amount of water suffer from drought conditions. This leads to the development of severe drought condition in that area. Consequently the herbivores such as humans and other animals suffer a lot resulting in famine condition. Such situation is spreading in several third world countries.

There are some plants that resist drought conditions. The property of resistance against water loss from the surface of plants is naturally checked by the plants. The special feature is controlled by a specific gene called ‘drought resistance (DR) gene’.

Plant biotechnology offers the introduction of DR gene from drought resistant plants into the staple crops for the benefit of enhanced crop yield.
The promising GM technology includes the use of techniques for increasing drought tolerance. Research works on these aspects are being investigated at the International Maize and Wheat Improvement Centre, Mexico and individual countries itself.

Wheat plants, that have been genetically modified to withstand drought, are now being tested in bio-safety greenhouse at the International Maize and Wheat Improvement Centre. Most of the plants produced have shown high tolerance to extreme low-water conditions. This research illustrates how recent advances in both molecular genetics and genetic engineering can be applied to enhance drought tolerance in plants. Progress h effects of drought on plants.

(c) The organisms selected for genome, projects were those that are mostly used in genetic and other scientific investigations. Thus, they may be regarded as model organisms. A model organism is an organism about which is a large amount of scientific knowledge is already available. These organisms include both prokaryotic and eukaryotic microorganisms as well as animals and plants. These organisms include E. coli, Bacillus subtilis, Archaeoglobus fulgidus, yeast {Saccharomyces cerevisiae), Arabidopsis thaliana, roundworm (Caenorhabditis elegans) and the fruitfly (Drosophila melanogaster). In addition, human genome project focussed on sequencing of the whole human genome.

E. coli is without any doubt, the best studied microorganism. Many of the technological tools available today were developed for E. coli genome project. The E. coli genome sequencing was completed in 1997. B. subtislis is a Gram-positive bacterium that colonizes leaf surfaces.

It is much used in industrial processes for both enzyme production and food supply fermentation. It is generally regarded as safe (GRAS).

S. cerevisiae is perhaps the most important fungal species used in biotechnological processes. Its name derives from the fact that it can ferment saccharose (sugar). S. cerevisiae genome sequencing project began in 1989, and the sequencing was completed in 1999. Arabidopsis thaliana is a small plant belonging to the Cruciferae family. It was recognized as a good organism for genetic studies in 1907, and has become a model plant for genetic studies.

Since it has the smallest genome among higher plants.

Further, its genome has a low amount of repetitive DNA. Arabidopsis genome sequencing project began in 1990 and was completed in 2000. The fruitfly, D. melanogaster, is fondly regarded the Queen of Genetics. Drosophila genome sequencing was completed in 2000. It has 180 x 106 bp and -16,000 genes.

Thus, the number of genes in Drosophila is less than four-times as many as in the bacterium E.coli.

Question 9.
(a) Mention the chief characteristics of stem cells. Give any two uses of such cells. [4]
(b) List the steps involved in the Sanger’s method for determining the amino acid sequence of proteins. State any one limitation of this method. [4]
(c) Name four centers or funding agencies which deal with Biotechnology and Bioinformatics in India. [2]
Answer:
(a) Embryonic stem cells : These are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocoel, a hollow cavity inside the blastocyst; and the inner cell mass, which is a group of cells at one end of the blastocoel that develop into the embryo proper.

Chief characteristics of stem cells : Embryonic stem cells differ from other kinds of cells in the body. Embryonic stem cells have three general properties:

• they are capable of dividing and renewing themselves for long periods,
• they are unspecialized; and
• they can give rise to specialized cell types.

Embryonic stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells which do not normally replicate themselves; Embryonic stem cells may replicate many times, or proliferate.

Embryonic stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry’ oxygen molecules through the bloodstream (like a red blood cell). Embryonic stem cells, can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

Uses of Embryonic stem cells: Studies of human embryonic stem cells will yield information about the complex events that occur during human development A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs.

Embryonic stem cells could also be used to test new drugs. For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Perhaps the most important potential application of human embryonic stem cells is the generation of cells and tissues that could be used for cell-based therapies.

(b) Protein sequencing is a technique to determine the amino acid sequence of a protein as well as which conformation the protein adopts and the extent to which it is complexed with any non-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understanding cellular processes. It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known. A protein sequencer is a machine that is used to determine the sequence of amino acids in a protein. They work by tagging and removing one amino acid at a time, which is analysed and identified. This is done repetitively for the whole polypeptide, until the whole sequence is established.

Determination of amino acid sequences :
Prior to amino acid sequence determination, it is essential to know the protein’s purity, molecular weight and its amino acid composition. A pure protein, if multimeric must be dissociated into its individual polypeptide chains and the molecular weight of the chains is determined. The molecular weight of a protein roughly tells about the number of amino acid residues contained in it. Each amino acid residue has an average molecular weight of 110 D. So if the molecular weight of a protein is 11000 D, we can predict that 100 amino acid residues are present. The amino acid composition tells the protein chemist about the strategies which could be employed in protein sequencing.

The following protein sequencing techniques are employed :
The first protein to be sequenced was the hormone ‘insulin’ whose deficiency leads to the disease diabetes. The Nobel Laureate, Fred Sanger invented a method of protein sequencing using a stepwise release and identification of amino acids starting from the N-terminal amino acid. The reagent employed by Sanger, known as fluoro-dinitro-benzene (Sanger’s reagent) reacted specifically with the free NH₂ group of the N-terminal amino acid and on acid hydrolysis, it yielded a yellow colored dinitro phenyl (DNP) derivative of the original N-terminal amino acid.

The polypeptide was subsequently, shortened by one amino acid. The DNP-amino acid was identified by comparison with other standard DNP amino acids using chromatography techniques. This procedure was repeated on the shortened polypeptide. In order to complete the sequence of insulin, Sanger used more than a gram of the hormone purified from a large number of pancreatic glands. However Sanger’s method has become obsolete and historical. The following steps summarise the Sanger’s method for determining the amino acid sequence.

• Hydrolysis by heating a sample of protein in 6M hydrochloric acid.
• Separation of polypeptide chains by ion – exchange chromatography.
• Fragmentation of the polypeptide chain by
  • Enzymatic methods using proteolytic enzyme, highly selective in hydrolyzing peptide bond,
  • Chemical methods using cyanogen bromide (CNBr) cleaves selectively the peptide bond following a methionine residue.
• Identification of N-terminal amino acid using Sanger’s reagent (fluoro-dinitrogen-benzene).
• Identification of DNP amino acid by comparison using chromatography by interpreting chromatogram.
• Repeating the procedure on the shortened polypeptide to identify rest of amino acids one by one.

Limitation : The method cannot be used in case the sample material is less in amount.

(c) (i) Institute (IARI) New-Delhi
(ii) National Diary Research Institute (NDRI) Kamal.
(iii) Indian Veterinary Research Institute (IVRI) Izat Nagar (Near Bareily U P.)
(iv) Centers for Plant Molecular Biology (CPMB) in various universities such as Delhi University, National Botanical Research Institute Lucknow.

ISC Class 12 Biotechnology Previous Year Question Papers

ISC Biotechnology Previous Year Question Paper 2014 Solved for Class 12

Maximum Marks: 80
Time allowed: Three hours

• Candidates are allowed additional 15 minutes for only reading the paper. They must NOT start writing during this time.
• Answer Question 1 (Compulsory) from Part I and five questions from Part II, choosing two questions from Section A, two questions from Section B and one question from either Section A or Section B.
• The intended marks for questions or parts of questions are given in brackets [ ].
• Transactions should be recorded in the answer book.
• All calculations should be shown clearly.
• All working, including rough work, should be done on the same page as, and adjacent to the rest of the answer.

Part-I
(Answer all questions)

Question 1.
(a) Mention any one significant difference between each of the following : [5]
(i) RNA polymerases and Tag DNA polymerases
(ii) ln-situ conservation and Ex-situ conservation
(iii) Micronutrients and Macronutrients
(iv) mRNA and tRNA
(v) Essential amino acids and Non-essential amino acids

(b) Answer the following questions : [5]
(i) Why is primer essential during DNA replication ?
(ii) Name a pest resistant crop developed using biotechnology techniques.
(iii) Why are glucose and lactose referred to as reducing sugars ?
(iv) What is the role of dcMTP during DNA sequencing ?
(v) How is the disease alkaptonuria caused ?

(c) Write the Ml form of the following: [5]
(i) BLAST
(ii) ESTS
(iii) MOD
(iv) RAM
(v) SNP

(d) Explain briefly: [5]
(i) Splicing
(ii) Mitotic arrest
(iii) Reverse transcription
(iv) Interferon
(v) Activation energy of enzymes
Answer:
(a) (i)RNA Polymerase: It is an enzyme found in both prokaryotic and eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of tmRNA.

Taq DNA Polymerase: It is a thermostable DNA polymerase, originally isolated from bacterium, Thermus aquaticus. It is used in polymerase chain reaction.

(ii) In-situ conservation: It means on-site conservation. It is the process of protecting an endangered plant or animal species in its natural habitat either by protecting or cleaning up the habitat itself, or by defending the species from predators.

Ex-situ conservation: It means literally, “off-site conservation”. It is the process of protecting an endangered species of plant or animal by removing part of the population from a threatened habitat and placing it in a new location, which may be a wild area or within the care of humans e.g., Zoos, Botanical garden, etc.

(iii) Micronutrients: Micronutrients are essential elements required by organisms in small quantities. They include microminerals and vitamins.

Macronutrients: Macronutrients include carbohydrates, proteins and fats required in larger quantities than micronutrients in the body

(iv) mRNA: mRNA is present in nucleus and functions in cytoplasm. It carries messages from DNA.

tRNA: tRNA is an adapter present in cytoplasm. It recognizes and brings amino acids near ribosomes for protein synthesis.

(v) Essential amino acid: The essential amino acids cannot be synthesized by the human body and are obtained from food.

Non-essential amino acid: The non-essential amino acids can be synthesized by the human body. They can be produced from other amino acids and substances in the diet and metabolism.

(b) (i) A primer is a strand of short RNA that functions as a starting point for DNA replication. It is required because the enzymes DNA polymerases that catalyze replication, can only add new nucleotides to an existing strand of DNA.

(ii) Bt crop is a pest resistant crop developed by using biotechnology techniques.

(iii) Glucose and lactose are the reducing sugars as they have an open chain with a free aldehyde or a ketone group.

(iv) ddNTP’s are a form of nucleotide that inhibit extension of the primer in DNA sequencing. Once a ddNTP has been incorporated into the DNA chain, it halts the process.

(v) Alkaptonuria (black mine disease or alcaptonuria): It is a rare, inherited recessive genetic disorder of tyrosine metabolism, caused due to mutation of gene.
Symptoms : Black urine, ochronosis, leading to osteoarthritis, kidney stones.

(c) (i) BLAST : Basic Local Alignment Search Tool

(ii) EST : Expressed Sequence Tag

(iii) MGD : Mouse Genome Database

(iv) RAM : Random Access Memory

(v) SNP : Single Nucleotide Polymorphism.

(d) (i) Splicing is the process by which introns(Non-coding) are removed from heteronuclear RNA to produce mature functional messanger RNA by uniting exons.

(ii) The process by which the mitotic cell cycle is stopped during one of the normal phases G1S, G2,M) of cell cycle.

(iii) It is the process by which DNA is synthesized from RNA template by reverse transcriptase enzyme. This process may be particularly helpful in treating diseases like HIV.

(iv) Interferon is a protein released by animal cells, generally in response to the presence of a virus. It inhibits virus replication.

(v) Activation energy of enzyme is the amount of energy required to bring substrates together to the point where they can react.

Part-II
(Answer any five questions)

Question 2.
(a) What are polysaccharides ? Name any three naturally occurring polysaccharides and give their structural units. [4]
(b) State one important use of each of the following in biotechnology : [4]
(i) Genomic DNA library and cDNA library
(ii) Transfection and Transformation
(c) What are single cell proteins ? [2]
Answer :
(a) Polysaccharides are the complex polymeric carbohydrate molecules composed of long chains of monosaccharide units linked together by glycosidic bonds. They vary in structure from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin. Starch, glycogen and cellulose are the natural polysaccharide and their structural units are glucose.

(b) (i) Genomic Library: This is a collection of clones that represent the complete genome of an organism. For construction of a Genomic library the entire genomic DNA is isolated from host cells or tissues, purified and broken randomly into fragments of correct size for cloning into a suitable vector.

The major use of Genomic library is hierarchichal shotgun sequencing.
cDNA Library : The library made from complementary or copy DNA (cDNA) is called cDNA library. The library represents the DNA of only eukaryotic organisms, not the prokaryotic once.

cDNA library’ is used to express eukaryotic gene in prokaryotes. cDNA libraries are most usefirl in reverse genetics where the additional genomic information is of less use. It is also useful for subsequently isolating the gene that codes for that mRNA.

(ii) Transformation : In the Biotechnology, Transformation means introduction of rDNA molecules into a living cell. It is the method of transfer of recombinants into the host cells. The DNA molecule comes in the contact of the cell surface and then it is taken up by the host cells.

Transfection : Transfection is the transfer of foreign DNA into cultural host cells mediated through chemicals. This method is used for the transfer of foreign DNA in the host cell. The recipient host cells are overlayed by this mixture. Consequently, the foreign DNA is taken up by the host cell.

(c) Single cell protein : The SCP is basically a non-pathogenic, fast growing microbial biomass rich in high quality of protein and can be commercially produced throughout the year and independent of climate (except algal process).
Example : Mushrooms and yeasts are good source of vitamin B complex.

Question 3.
(a) Name any four vectors used in gene cloning technique. Also, state the unique properties of each of them. [4]
(b) With reference to tissue culture, explain the importance of: [4]
(i) Triploid plants
(ii) Haploid plants
(c) Give two uses of site directed mutagenesis. [2]
Answer:
(a) Vectors are tools used by molecular biologists to insert genes or pieces of foreign DNA into host cells. The four main types of vector commonly used include plasmids, bacteriophages, cosmids and yeast artificial chromosomes (YAC’s).

Plasmid: It is an extra chromosomal circular DNA molecule that independently replicates inside the bacterial cell and some yeasts; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb).

Phage : Designed bacteriophage lambda (λ) and M13 ; linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb. M13 is a filamentous phage which infects E-coli. cloning limit: 10 kb.

Cosmids: A constructed extrachromosomal circular DNA molecule that combines features of plasmids and (cos) sites of phage; cloning limit: 45 kb.

Yeast Artificial Chromosomes (YAC): An artificial chromosome that contains telomeres, origin of replication, a yeast centromere, restriction enzymes sites and a selectable marker for identification in yeast cells ; cloning limit: 1Mb.

(b) (i) Applications / Importances of triploid plants :

• Autotriploidy induces gigas effect i.e., large-sized leaves, flowers, fruits and seeds.
• Autotriploids are sterile (with faulty pairing and distribution of chromosomes and abnormal gametes) and reproduce vegetatively only. They are useful for the production of seedless plants.

(ii) Applications / Importances of haploid plants :

• Haploid plants have been used in breeding programmes of rice, wheat, barley, Brassicaspps, tobacco, potato, etc.
• A doubled haploid strain are produced in only two years a net saving of 4 years for the production of homozygous lines by selfing or close breeding of both self and cross pollinated crops. They may be released directly as a variety if it performs well in yield trials.
• Haploid plants are most suitable for the study of recessive mutations.

(c) Uses of Site-directed mutagenesis

• To study the protein structure, gene expression, gene regulation and function relationships.
• To make specific and intentional changes to the DNA sequence of a gene and any gene products.

Question 4.
(a) Discuss the following innovations in biotechnology : [4]
(i) Recombinant hepatitis B vaccine
(ii) Tomato fruit with delayed ripening
(b) Explain the method used for DNA sequencing by chemical degradation technique. [4]
(c) Mention the principle involved in the freeze preservation of germplasm. [2]
Answer:
(a) (i) In July of 1986, the food and drug administration (FDA) approved the first genetically engineered vaccine for humans : Hepatitis B vaccine. Hepatitis B vaccine is developed for the prevention of hepatitis B virus infection. A microorganism, e.g., yeast for expression of hepatitis B surface antigen (HBsAg) is used as vaccine against hepatitis B virus.

The vaccine contains one of the viral envelop proteins (HBsAg). The gene coding this protein is identified and integrated into a suitable expression vector and introduced into a suitable host where the protein is produced in large quantity. Protein is then isolated and purified from host cell and used in preparation of vaccine.

(ii) A plant with delayed fruit ripening
‘Flavr Savr’ variety of tomato:
In transgenic tomato, the expression of gene for the production of polygalacturonase was blocked as this enzyme degrade pectin. In the absence of enzyme, pectin degradation is stopped and the fruit remains fresh for long time. It retains flavour, has superior taste and higher quantity’ of total soluble solids.

(b) The structure of DNA (e.g., gene insert, a recombinant plasmid or entire genome) can be analysed by determining the nucleotide sequences. In molecular cloning, the information of nucleotide sequences are essential. In 1965, Robert Holley and his research group at Cornell University completely sequenced nucleotides of tRNAala (tRNA for yeast alanine). In 1977, the following two methods were developed.

Allan Maxam and Walter Gilbert developed a chemical method of DNA sequencing. In this method, end-labelled DNA is subjected to base specific cleavage reaction before gel separation. In routine sequencing of DNA this method is not commonly followed. In the same year (1977) Frederick Sanger and co-workers developed an enzymatic method of DNA sequencing. It is also called dideoxynucleotide chain termination method because dideoxynucleotides are used as chain terminator to produce a ladder of molecules.

Maxam and Gilbert’s Chemical Degradation Method
In this method, DNA sequencing involves the following steps :

• Labelling of 3 ’ends of DNA with isotopic phosphorus (32P).
• Separation of two strands labelled at 3 ’ ends.
• Separation of mixture in four sets, each treated with a different reagent which can degrade only G or C, or A and G or T and C.
• Electrophoretic separation of each sample in four different gel.
• Autoradiography of gels and determination of the sequence from position of bands in four lanes of gel.

(c) Freezing: When cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or -196°C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped.

Storage : The frozen cells and tissues are stored in a liquid nitrogen refrigerator, the temperature must not be rise above -130°C otherwise ice crystals may be formed.

Thawing: Thawing of the frozen materials is achieved by plunging the vials into water at 37-40°C (thawing rate of 500°-700°C/min) for 90 seconds. The material is then transferred into an ice bath till it is recultured.

Reculture : Materials subjected to cryopreservation may show some special requirements during reculture. For example, shoot-tips preserved seedlings of tomato required GA3 for developing into shoots. Similarly survival of carrot plantlets was generally improved by activated charcoal.

Question 5.
(a) Explain the principle and any two uses of each of the following biochemical techniques : [4]
(i) Partition Chromatography .
(ii) X-Ray Crystallography.
(b) Enumerate the steps involved in Southern blotting technique. Also, write two applications of this technique. [4]
(c) Mention any two methods used in the isolation of single cells from plant organs. [2]
Answer:
(a) (i) Partition Chromatography: Partition chromatography is the process of separation whereby the components of the mixture get distributed into two liquid phases due to differences in partition coefficients during the flow of mobile phase in the chromatography column.

Here the molecules get preferential separation in between two phases, i.e., both stationary phase and mobile phase are liquid in nature. So molecules get dispersed into either phases preferentially. Polar molecules get partitioned into polar phase and vice-versa. This mode of partition chromatography applies to liquid-liquid, liquid-gas chromatography and not to solid-gas chromatography. Because partition is the phenomenon in between a liquid and liquid or liquid and gas or gas and gas. But not in solid involvement.
Uses:

• It is used to separate the amino acids.
• It is extensively used for the study of lipid-soluble substances.

(ii) The technique of single-crystal X-ray crystallography has three basic steps :
The first and often most difficult step is to obtain an adequate crystal of the material under study. The crystal should be sufficiently large (typically larger than 100 microns in all dimensions), pure in composition and regular in structure.

In the second step, the crystal is placed in an intense beam of X-rays, usually of a . single wavelength (monochromatic X-rays), producing the regular pattern of reflections.

As the crystal is gradually rotated, previous reflections disappear and new ones appear; the intensity of every spot is recorded at every orientation of the crystal. Multiple data sets may have to be collected, with each set covering slightly more than half a lull rotation of the crystal and typically containing tens of thousands of reflection intensities.

In the third step, these data combined computationally with complementary chemical information to produce and refine a model of the arrangement of atoms within the crystal. The final, refined model of the atomic arrangement now called a crystal structure is usually stored in a public database.

Uses:

• It is used to determine the atomic and molecular structure of a crystal.
• It is also used in X-ray diffraction to rotate the samples.

(b) Southern blotting is a technique for transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon membrane, and is carried out prior to detection of specific molecules by hybridization probing.

In this technique, DNA is usually converted into conveniently sized fragments by restriction digestion and separated by gel electrophoresis, usually on an agarose gel. The DNA is denatured into single strands by incubation by alkali treatment.

The DNA is transferred to a nitrocellulose filter membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation they had on the gel. This process is called blotting.

The nitrocellulose membrane is now: removed from the blotting stack.

The blot is incubated with many copies of a radioactive probe which is single-stranded DNA. This probe detect and identify base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g., alkaline phosphatase or horseradish peroxidase). This step is known as hybridization reaction.

The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts to a coloured product that can be seen or gives off light which will expose X-ray film. If the probe was labelled with radioactivity, it can expose X-ray film directly. The images of radioactive probe are revealed as distinct bands on the developed X-ray film.

Applications of Southern blotting

• It is used in DNA fingerprinting.
• Identify mutation, deletion and gene rearrangement.
• Prognosis of cancer and prenatal diagnosis of genetic disease.

(c) Methods of single cell isolation from plant organ: Leaf mesophyll tissue and callus are the most suitable materials to isolate a single cell. Leaf mesophyll tissue contains a homogeneous population of cells. There are two methods described for isolation of a single cell:

Mechanical Method: Mechanical isolation involves tearing of surface sterilized explants to expose the cells followed by scrapping of the cells with a fine scalpel to liberate the single cells without any damage. These cells are dipped in liquid medium.
About 10 grams of leaves are macerated in 40 ml of buffered medium using pestle and mortar. The homogenate is filtered through muslin cloth. Cells are w ashed by centrifugation at low speed. Cells are collected and debris are removed.

Enzymatic Method : Using this method, maximum amount of cells can be isolated with minimum damage and injury in the cells. It is accomplished by providing osmotic protection to the cells. The enzyme (pectinase/macerozyme) degrades the middle lamella and cell walls of the parenchy matous tissue as a result, individual cells are set free. Osmotic protection is provided to the cells while the enzyme macerozyme degrades the middle lamella and cell w all of the paranchymatous tissues.

Question 6.
(a) State the role of any four enzymes involved in the process of DNA replication. [4]
(b ) Outline the basic steps involved in the in vitro regeneration of a complete plant from a single cell. [4]
(c) Name any two biochemical techniques based on solubility. [2]
Answer:
(a) Enzymes taking part in Replication
Four different enzymes have been reported which are given below :

Exonuclease : This enzyme breaks down the hydrogen bonds coupling nitrogenous bases in DNA molecules so that both the chains get apart during replication.

Endonuclease: This enzyme breaks down the chain or chains of DNA molecule. Whenever it appears, it results in dispersive type of replication.

Polymerase or replicase enzyme: It catalyses the formation of one polynucleotide chain that is a copy of another (complementary chain). This new chain is synthesized from the raw material already present in nucleoplasm.

Ligase: This enzyme is used in the formation of bonds between the nitrogenous bases such as A = T and C = G. Whenever the appearance of the enzyme is delayed, conservative type of replication results.

(b) Steps involved in in vitro regeneration of a complete plants.
Regeneration refers to the development of organised structures like roots, shoots, flower buds, somatic embryos (SE’s), etc. from cultured cells/tissues; the term organogenesis is also used to describe these events. Root regeneration occurs quite frequently, but it is useful only in case of shoots and embryo germination. Only shoot and SE regeneration’s give rise to complete plants, which is essential for applications of tissue culture technology in agriculture and horticulture. Regeneration may occur either directly from the explant or may follow an intervening callus phase.

Basic technique of Plant Tissue Culture: The basic technique of plant tissue culture involves the following steps :
Preparation and Sterilisation of Suitable Nutrient Medium: Suitable nutrient medium as per objective of culture is prepared and transferred into suitable containers. Culture- medium is rich in sucrose, minerals, vitamins, and hormones. Yeast extract, coconut milk, are also added. The culture is completely sterilized in an autoclave.

Selection of explants : Selection of explants such as shoot tip should be done.

Sterilisation of explants: Surface sterilisation of the explants by disinfectants (eg., sodium hypochlorite or mercuric chloride) and then washing the explants with sterile distilled water is essential.

Inoculation: Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilised by autoclaving to avoid microbial contamination) in culture vessels under sterile conditions is done.

Incubations : Growing the culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e., artificial light; 16 hours of photoperiod), temperature (- 26°C) and relative humidity (50-60%) is required.

Regeneration : An unorganized mass of cells developing from explants is called callus. The callus gives rise to embryoids which can develop into whole plant if the medium is provided with proper concentration of hormones. This property of developing every somatic cell into a full fledged plant is called totipotency. Regeneration of plants from cultured plant tissues is carried out.

Hardening: Hardening is gradual exposure of plantlets to an environmental conditions.

Plantlet transfer : After hardening, plantlets are transferred to the greenhouse or field conditions following acclimatization (hardening) of regenerated plants.

(c) Biochemical techniques based on solubility :

• Salt precipitation
• Precipitation with organic solvent.

Question 7.
(a) Explain the operon concept of gene regulation with the help of an example. [4]
(b) Enumerate the secondary and tertiary structures of proteins. Also, mention any two important functions of proteins. [4]
(c) Mention any two potential uses of cloned stem cells. [2]
Answer:
(a) Operon is a regulatory gene system, often encoding a coordinated group of enzymes involved in a usually by interaction with and inactivation of a repressor, of trancription of the genes encoding the enzymes. Lac-operon is type of inducible pathway as the operon is switched on in response to the presence of chemical (lactose, a subtrate) called Inducer, which after coming in contact with repressor, form a non-DNA binding complex so as to free the operator gene for transcription. The enzymes formed in response to the presence of its subtrate are called Inducible enzymes.
Lac-operon of E.coli has following components.

• There are three structural genes Z, Y, A, which transcribe polycistronic /mRNA.
• Promoter gene is the site where RNA polymerase binds for transcription.
• Operator gene function as a switch for the operon. In absence of lactose, repressor binds to operator gene and the RNA polymerase cannot move from promoter gene and thus there is no transcription.
• i-gene is a regulator gene and synthesises a repressor protein. In the presence of lactose (inducer), the repressor bind to it and become inactive repressor, which does not bind to operator gene and RNA polymerase from promoter gene move to structural genes to help transcription.

Example: The lactose operon is an example of negative regulation. When E. coli cells are growing in a medium containing lactose as the only carbon source, some of the lactose is converted to allolactose. Allolactose acts as an inducer and turns on the lac-operon. In the presence of glucose and the absence of lactose, allolactose is not produced and the lac operon is turned off. Since glucose is a readily available energy source, cells won’t use lactose until they consume all the glucose. When both glucose and lactose are present in the medium, the operon is also turned off. In this case, the operon is regulated by catabolite repression.

(b) Secondary Structure (2° structure) : It is development of new stearic relationships amongst the amino acids for protecting their peptide bonds through formation of intrapolypeptide and interpolypeptide hydrogen bonds. Secondary structure is of three types—a-helix, p-pleated and collagen helix. The prefixes a and p signify the first and second types of secondary structure discovered by Pauling and Corey (1951).

α-Helix: The polypeptide chain is spirally coiled, generally in a clockwise or right-handed fashion (Fig.). There are 3.6 amino acid residues per turn of the spiral. The spiral is stabilized by straight hydrogen bonds between imide group (-NH-) of one amino acid and carbonyl group (—CO—) of fourth amino acid residue. In this way, all the imide and carbonyl groups become hydrogen bonded. R-groups occur towards the outer side of a-helix. a-helix is the final structure in certain fibrous proteins, e.g., keratin (hair, nail, horn), epidermis (skin).

β-Pleated Sheets : Two or more polypeptide chains come together and form a sheet. Condensation is little. However, twisting does occur. The same polypeptide may fold over itself to form two strands for β-pleating. Adjacent polypeptide chains may occur in parallel (e.g., p-keratin) or anti-parallel (e.g., silk fibrin). Straight hydrogen bonds occur between imide (-NH-) group of one polypeptide and carbonyl (-CO-) group of adjacent polypeptide. Cross-linkages help in stabilization of β-pleated sheets.

Collagen Helix : Collagen has a large amount of glycine (25%) and proline (and hydroxyproline, 25%). It cannot form a-helix due to them. Three of its polypeptide each having about 1000 amino acid residues, come together with each forming an extended left-handed helix. They run parallel, form a right-handed super-helix that is stabilised by hydrogen bonds amongst the three. The triple helix of collagen is often called tropo- collagen. Its one end is stabilised by -S-S- linkages amongst the three chains. Collagen occurs in those tissues where extensibility is limited, e.g., connective tissue, tendons, bones.

Tertiary Structure (3° Structure) :
Tertiary (3°) structure is bending and folding of secondary strand (2°) of polypeptide (a-helix or β-pleated sheet) in such a way as to form a compact structure with functional sites being established over its surface by coming together of polar regions of specific amino acids. Hydrophobic parts of amino acids generally pass into the interior of protein. When both a-helix and β-pleating occur in the same polypeptide, tertiary structure segregates them into specific areas. Tertiary structure is stabilised by five types of bonds :

1. Disulphide Bonds : Between hydrogen sulphide groups of two cysteine or methionine amino acids.
2. Hydrogen Bonds : They occur between hydrogen and oxygen atoms of various groups like -NH OC- or -OH OC-.
3. Ionic Bonds : They appear between oppositely charged ionised groups of two amino acids, e.g., NH3+ and -COO .
4. Hydrophobic Interactions: They are not true bonds but involve coming together of non-polar R-groups of two amino acids. Hydrophobic interactions are important in compaction of protein as they exclude water molecules in the area of their occurrence.
5. van derWaals Interactions : They develop between two closely placed polar groups through charge fluctuations between the two.

Functions of Proteins:

• Some proteins as hormones regulate many body functions. For example, the hormone insulin is a protein. It regulates sugar level in the blood.
• Some proteins as enzymes catalyse or help in biochemical reactions. For example, pepsin and trypsin.

(c) (i) Stem cells can be modified to bone, muscle, cartilage and other specialized types of cells, they have the potential to treat many diseases, including Parkinson’s, Alzheimer’s, diabetes and cancer.
(ii) New medications could be tested for safety on specialized cells generated in large numbers from stem cell lines.

Question 8.
(a) What is bioinformatics ? Mention its significant applications.
(b) Name the first bio technologically created mammalian clone and also explain the methodology involved in it.
(c) Define the term semi-gamy.
Answer:
(a) Bioinformatics is the application of computer technology to the management of biological information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied to gene-based drug discovery and development. Bioinformatics uses many areas of computer science, statistics, mathematics and engineering to process biological data.

Applications of Bioinformatics:

• It is used to search the genome of thousands of organisms, containing billions of nucleotides. These programs would compensate for mutations (exchanged, deleted or inserted bases) in the DNA sequence, in order to identify sequences that are related, but not identical.
• It is used for measuring biodiversity.
• The expression of many genes can be determined by this process.
• Bioinformatic techniques have been applied to explore various steps in the analysis of regulation.
• In order to trace the homology of newly identified protein.

(b) Dolly (sheep) is the first cloned sheep. The methods involved in creation of dolly are :
To Isolate donor nucleus: Firstly isolate the nucleus from a somatic (non-reproductive) cell of a adult donor sheep. The nucleus contains the complete genetic material of the organism. A micropipette is used to capture the nucleus and remove it from the cell.

Recovery of unfertilized eggs: Remove some unfertilized egg cells (reproductive) from a female sheep. Many eggs are needed since not all of them will survive the various steps of cloning.

Remove the egg’s nucleus: Nucleus is removed from egg which contains only one-half of the sheep’s genetic material.

Insert donor nucleus : Insert the nucleus, with its complete genetic material, isolated from the donor mammal in Step 1 into the egg cell that has no nuclear material. The egg’s genetic material now contains all traits from the donor adult. This egg is genetically identical to the donor adult.

Place the egg into womb : Transplant the eggs into a female sheep’s womb. Those eggs that survive will continue to develop into embryos. The egg matures in the womb. The offspring dolly is born, a clone (genetically identical) of the donor sheep.

After cloning was successfully demonstrated through the production of Dolly, many other large mammals have been cloned, including horses and bulls. The attempt to clone argali (mountain sheep) did not produce viable embryos. The reprogramming process cells need to go through during cloning is not perfect and embryos produced by nuclear transfer often show abnormal development.

(c) Semigamy: It is a type of facultative apomix in which the male sperm nucleus does not fuse with the egg nucleus after penetrating the egg in the embryo sac. Subsequent development can give rise to an embryo containing haploid chimeral tissues of paternal and maternal origins. In cotton, the semigametic phenomena was first reported by Turcotte and Feaster (1963), who developed the Pimaline 57-4 that produced haploid seeds at a high frequency.

Question 9.
(a) Write short notes on : [4]
(i) Protein databases
(ii) Hardware and Software of computers
(b) Name any four resources obtained from NCBI and give their uses. [4]
(c) Mention any two types of sequence analysis used in bioinformatics. [2]
Answer:
(a) (i) Protein Databases: The protein database is a collection of sequences from several sources, including translations from annotated coding regions in GenBank, RefSeq and TPA, as well as records from SwissProt, PIR, PRF, and PDB. Protein sequences are the fundamental determinants of biological structure and function. Protein databases are more specialized than primary sequence databases. They contain information derived from the primary sequence databases.
Example : PIR, TIGR, PFam, PRINTS, ProDom, ProSite, ALIGN, SWISS-PROT & TREMBL, etc.

(ii) Hardware and Software of computers
Hardware: Hardware is the collection of physical components that constitutes a computer system including peripherals such as the monitor, mouse, keyboard, computer data storage, hard drive disk (HDD), system unit (graphic cards, sound cards, memory motherboard and chips), etc., all of which are physical objects that can be touched. In contrast, software is instructions that can be stored and ran by hardware.

Software: Written programs or procedures or rules and associated documentation pertaining to the operation of a computer system and that are stored in read/write memory. Bases on their goals, they could be Application software, System software, Malicious software or malware, etc.

(b) The NCBI located at National library of Medicine was established in Bethesda, Maryland in 1988 through legislation sponsored by Senator Claude Pepper.
Useful resources:

• Genome sequencing data in GeneBank.
• An index of biomedical research articles in PubMed Central and PubMed.
• Entrez search engine, used to access literature (abstracts), sequence and structure databases.

(c) PDB (Protein Data Bank): This database has sequence of those protein whose 3 -D structures are known.
Source : NCBI-U.S.A.; EBI, UK.
Blast (Basic Local Alignment Search Tool): Blast is a family of user-friendly sequence similarity search tools on the web. The Blast server is supported through NCBI (National Centre for Biotechnology Information) U.S.A. This tool is designed to identify potential homologues for a given sequence. It can analyse both DNA and protein sequences. A local alignment finds the optimal alignment between subregions or local regions of specified sequences.

ISC Class 12 Biotechnology Previous Year Question Papers

ISC Biotechnology Previous Year Question Paper 2016 Solved for Class 12

Maximum Marks: 80
Time allowed: Three hours

• Candidates are allowed additional 15 minutes for only reading the paper. They must NOT start writing during this time.
• Answer Question 1 (Compulsory) from Part I and five questions from Part II, choosing two questions from Section A, two questions from Section B and one question from either Section A or Section B.
• The intended marks for questions or parts of questions are given in brackets [ ].
• Transactions should be recorded in the answer book.
• All calculations should be shown clearly.
• All working, including rough work, should be done on the same page as, and adjacent to the rest of the answer.

Part-I
(Answer all questions)

Question 1.
(a) Mention any one significant difference between each of the following : [5]
(i) Ligases and Helicases
(ii) Introns and Exons
(iii) Gel electrophoresis and Gel permeation
(iv) Sucrose and Starch
(v) Plasmids and Phages

(b) Answer the following questions : [5]
(i) Why is the nutrient medium autoclaved before using it for cell culture?
(ii) Name the enzyme that can synthesize DNA at a high temperature.
(iii) Why are restriction enzymes also called as molecular scissors ?
(iv) Name the nitrogenous bases present in RNA.
(v) Why is Agrobacterium tumifaciens called as the natural genetic engineer of plants ?

(c) Write the full form of each of the following :
(i) YAC
(ii) NCBI
(iii) RAM
(iv) SNP
(v) EMBL

(d) Explain briefly : [5]
(i) Somatic hybridization
(ii) Promoter gene
(iii) Site directed mutagenesis
(iv) DNA probes
(v) Primer
Answer:
(a) (i) Ligases are the enzymes which help in linking up of okazaki (DNA) segments produced on the parent strand with 5′-3′ direction. Hclicases help in unwinding the DNA helix, using ATP hydrolysis as a source of energy.

(ii) Introns are the intervening sequences which do not appear in the mature or processed RNA.
Exons are the coding sequences or expressed sequences that form functional and processed RNA.

(iii) Gel Electrophoresis is a technique by which negatively charged DNA fragments are separated by forcing them to move towards the anode under an electric field through a medium/matrix.

Gel permeation or filtration involves that molecules of different sizes can be separated from each other on the basis of their ability to enter the pores within the beaded gel, followed by passing down a column containing the gel. The technique is used in protein purification.

(iv) Sucrose is a disaccharide formed of glucose and fructose molecules while starch is a polysaccharide molecules formed of large number of glucose molecules arranged in the form of chains.

(v) Plasmids are the extra-chromosomal, self replicating, circular, double stranded DNA molecules present in bacteria.
Phages are the viruses which infect bacteria/cell. lyse it, integrate its DNA into it and replicate with the host chromosome.

(b) (i) The nutrient medium is autoclaved to make it sterilise i.e., free from microbes.

(ii) DNA polymerase isolated from a bacterium Thermus aquations.

(iii) Restriction enzymes are called ‘molecular scissors’ because they make cuts at specific positions/recognition sites within both strands of DNA.

(iv) Adenine, guanine, uracil, cytosine.

(v) Agrobacterium tumefaciens is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into tumour cells. By manipulating its Ti plasmid, it has now been modified into a useful cloning vector for delivering gene of our interest into a variety of plants.

(c) (i) YAC = Yeast Artificial Chromosome.

(ii) NCBf = National Centre for Bioinformatics Information.

(iii) RAM = Random Access Memory.

(iv) SNP = Short Nucleotide Polymorphism.

(v) EMBL = European Molecular Biology Laboratory.

(d) (i) Somatic hybrids are the hybrid plants produced through the fusion of protoplasts from two different varieties of plants each haring a desirable character. The hybrid protoplasts can be further grown to form a new plant.

(ii) Promoter gene a gene haring a regulatory sequence of DNA that initiates the expression of a gene.

(iii) Site directed mutagenesis involves induction of specified or desired changes in the base sequence at specified sites of genes, most successfully achieved by overlap extension PCR.
Or
The process of nucleotide changes in the cloned genes by specific in mutagenesis.

(iv) DNA probes are short 15-30 bases long, labelled oligonucleotides (RNA – DNA) used to detect complementary nucleotide sequences after hybridization and the auto radiograms gives many bands of different sizes.

(v) Primer is a short oligonucleotide that hybridizes with the template strand and gives a 3′- OH end at which a DNA polymerase start synthesis of DNA chain.

Part-II
(Answer any five questions)

Question 2.
(a) With reference to amino acids, explain :
(i) Any one physical and any one chemical property of amino acids.
(ii) Essential and non-essential amino acids.
(b) Briefly outline the various steps involved in the gene cloning technique
(c) List any four characteristics of genetic code.
Answer:
(a) They are the building blocks of molecular proteins.
(i) Amino acids are the organic acids (with carboxylic group-COOH) having amino group (-NH₂) generally attached to a-carbon or carbon next to the carboxylic group. Amino acids condenses to produce peptide (-NHCO-) bond.

(ii) Essential amino acids are essential for our body but they are not synthesised inside our body e.g., valine, isoleucine, lysine etc. They are supplemented through diet. Non-essential amino acids are those which are synthesised through transformation and transamination inside our body e.g., serine, alanine etc.

(b) The various steps involved in gene cloning technique are :

• Identification and isolation of desired DNA. .
• Amplification of gene of interest using PCR.
• Fragmentation/cutting of desired DNA and vector DNA by restriction enzymes.
• Ligation/joining of desired DNA fragment into vector using ligase enzyme.
• Transferring the recombinant DNA into host cell/organism by transformation, transfection, electroporation, microinjection (vectorless transfer), particle bombardment gun (Biolistics) (Vectorless transfer) or by Agrobacterium/retrovirus mediated gene transfer..
• Culturing the host cell for obtaining the foreign gene product/recombinant protein.
• Extraction of desired products or downstream processing.

(c) The characteristics of genetic codes are :

• Codes are universal.
• Codes are unambiguous and specific.
• Code is degenerate i.e., some amino acids are coded by more than one codon.
• Codons are read in a continuous fashion i.e., there are no commas or punctuation’s.

Question 3.
(a) Describe the three dimensional structure of DNA as proposed by Watson, Crick and Wilkins. Name a biochemical technique that was used by them to confirm the structure of DNA. [4]
(b) Explain the role of the following enzymes during the process of protein synthesis : [4]
(i) RNA polymerases and amino acyl tRNA synthetase.
(ii) Start codons and end codons.
(c) Why are auxins and cytokinins used in plant tissue culture ? [2]
Answer:
(a) Watson, Crick and Wilkins described the structure of DNA but also indicated how it could be replicated and transfer from one organism to its off spring.
Salient features of double helix structure of DNA include :

• Double helix made of two polynucleotide chains.
• Sugar and phosphate form backbone and N-base project inside.
• The two chains are antiparallel with one chain has the polarity of 5′ → 3′, the other has 3’→ 5.
• Presence of double hydrogen bonds between A=T, and triple bond to between G = C The . purine always comes opposite to pyrimidines to create uniform distance.
• Two chains are coiled in a right handed fashion.
• Pitch (one turn) of helix is 3.4 nm (= 34 A) contains 10 bp with 0.34 nm (3.4 A) gap between adjacent bps. (1 nm = 10-9 m)
• Two chains of DNA are 20 A far apart, due to the pairing of purines with pyrimidines. This distance remains constant.
• The plane of one base pair stacks over the other in double helix to confer stability

Rosalind Franklin confirmed this structure of DNA through X-Ray crystallography.

(b) (i) RNA polymerizes get associated transiently with initiation sigma factor (σ). They do not require a primer and can initiate the synthesis of a new chain on the template strand. It binds to the promoter site to start synthesis. The first nucleotide is labelled as + 1 and is called the transcription start site. It polymerizes ribonucleotides in 5′ → 3′ direction over 3′ → 5′ template DNA.

In prokaryotes, a single RNA polymerase transcribes all m-RNA, r-RNA and t-RNA. However, in case of Eukaryotas there are three different polymerizes i.e., RP+1 for r-RNA, RP-II for m-RNA and RP-III for t-RNA.
Amino acyl t-RNA syntheses couple each amino acid to its appropriate set of t-RNA molecules. There are 20 synthesizes for each of the 20 natural amino acids.

(ii) In prokaryotes, AUG acts as a start/initiation codon as well as codes for methionine. GUG acts as start codon for valine in Eukaryotes. UAA, UAQ UGA function as stop codons and do not code for any amino acid.

(c) Role of auxin :

• It helps in the formation of callus and in the development of xylem along with the promotion of cambial activity.
• It is used for the elongation and enlargement of plant cell and it inhibits the promotion of growths of apical and lateral buds.

Role of cytokinin:

• It stimulates the cell division process.
• It helps in the morphogenesis of plant cell, along with auxins.

Question 4.
(a) Explain how DNA technology has been used to create the following : [4]
(i) Dolly
(ii) Hepatitis B vaccine
(b) Write short notes on : [4]
(i) Batch culture and continuous culture.
(ii) Salinity resistance in crops.
(c) Name any two chemicals used to prepare the gel for gel electrophoresis. [2]
Answer:
(a) (i) Dolly – The first cloned animal
Using nuclear transfer technique, the world’s first mammalian clone – Dolly, was born in February 1996. In 1995, Ian Wilmut and his research group (Scotland) took out udder from a six year old sheep A called clone mother, and put in a special solution. Nucleus of udder cell was taken out and put in a solution. At the same time an unfertilized egg was taken out from another sheep B called egg mother. Nucleus of the egg was removed and nucleated egg was put in a culture medium. The nucleus of udder cell and nucleated egg cell were put together followed by mild electric shock. Consequently nucleus was taken up by the nucleated cell. This cell was incubated onto growth medium then transferred into a surrogate mother. A little lamb Dolly was born in February, 1996.

(ii) It is true that proteins (vaccines) stimulate immune system and cause to secrete specific antibodies. Such specific amino acid sequences in the protein that stimulate immune response are called epitopes. Based on selected epitopes recombinant vaccines may be produced on commercial level which can prove more effective and safer than the conventional vaccines.

Working on these lines, a recombinant Hepatitis B vaccine was produced by cloning the synthetic gene (for the surface antigen of the virus) in yeast cells. This gene expressed well in yeast cells and produced 22 nm particles of hepatitis B virus (HBV) surface antigen (as produced in patients) infected with hepatitis B virus. The recombinant vaccine has high immunogenecity. This product has been marketed as a vaccine for protection against HBV infection.

(b) Tissues and cells cultured in a liquid medium produce a suspension of single cells and cells clumps of few to many cells, are called suspension culture. Suspension cultures are of two types:

(i) Batch culture: In a batch culture, the same medium and all the cells produced are retained in the culture vessel e.g., culture flasks (100-250 ml), fermenters (variable size) etc. The cell number of biomass, of a batch culture exhibits a typical sigmoid curve represented by lag phase (cell number or biomass remain unchanged), log phase (rapid increase in cell number) and finally stationary phase (cell member does not change). The lag-phase lasts about 3-4 cell generation. The stationary phase is forced on the culture.

• due to depletion of nutrients
• and due to accumulation of wastes.

They are maintained by subculturing. They are unsuitable for studies on cell growth and metabolism because of constant change in cell density and nutritional status.

Continuous culture: Here the cell population is maintained in a steady state by regularly replacing a portion of used or spend medium. Such culture systems are either

• closed or
• open system.

In closed system, cells are separated from the used medium, taken out for replacement and added back to the culture so that biomass keeps on increasing. In contrast, both cells and used medium are taken out from open continuous system and replaced by equal volumes of fresh medium.

(ii) A plant which bears a foreign gene for desired function of other organism is called transgenic crop. Using biotechnological approaches stress/salinity tolerant plants can be produced. They secrete stress-related osmolytes such as sugars (fructans and trehalose) sugar alcohols (mannitol) amino acids (betaine, glycine and proline) and other proteins. For example, betaine is highly effective osmolyte that accumulates in some plants during, water stress or high salinity: Betaine is synthesised both in bacteria and plants. A transgenic tobacco was prepared by transferring E.coli bet A. gene through Ti-plasmid. The transgenic tobacco was 80% salt tolerant (i.e. 300 mM) than the normal tobacco.

(c) (i) Agarose gels
(ii) Polyacrylamide gels.

Question 5.
(a) Give the step wise procedure of the Southern Blotting technique. Mention any two differences
between Southern Blotting technique and Northern Blotting technique. [4]
(b) Explain the principle and any two applications of each of the following biochemical techniques:
(i) Ion Exchange Chromatography
(ii) Colorimetry [4]
(c) What is the cause of inborn metabolic disorders? Give any two examples of these disorders. [2]
Answer:
(a) Southern Blotting (Hybridization) Technique : In 1975, Edward M. Southern developed the technique of DNA separation and its hybridization. Therefore, in his honor this technique is known as ‘Southern blotting or Southern hybridization technique’. A specific DNA fragment can be separated and identified in a heterologous population of DNA molecules on the basis of binding of DNA probe with its complementary DNA strand.

The genomic DNA is isolated from the clone and digested with restriction enzymes. The DNA fragments are separated by agarose gel electrophoresis. Different DNA bands are formed on agarose gel which represents DNA fragments of varying sizes. These fragments are transferred from gel to nylon or nitrocellulose membrane. The process of DNA transfer is called ‘blotting’.

A nitrocellulose membrane is put over the gel. Many layers of filter paper are placed over nitrocellulose membrane. This assembly is put in a container having NaOH solution. NaOH denatures DNA and results in formation of single stranded DNA. DNA fragments are transferred from gel to membrane by capillary action.

The DNA fragments are fixed to membrane by using UV radiation or baking at 80°C. The pattern of DNA bands on membrane corresponds to the position of DNA on gel. The membrane is put in solution containing radio labelled DNA probe and incubated for some time. DNA probe hybridizes complementary DNA fragments fixed on membrane. It is gently washed at 12°C and dried. The membrane is exposed through a photographic film. DNA bands formed on photographic film corresponds to the original position of DNA fragments present on agarose gel.

Southern Blotting technique involves separation and identification of a specific DNA fragment. In Northern Blotting technique the RNA is analysed rather than DNA. During southern blotting NaOH denatures DNA to form single stranded DNA which are transferred from gel to nitro cellulose membrane. In northern blotting total RNA molecules are extracted and then mRNA molecules are isolated by using oligo (dT) cellulose Chromatography. RNA samples separated are transferred to a nylon membrane.

(b) (i) Ion Exchange Chromatography Principle: It is based on reversible exchange of ions in solution with ions electrostatically bound to some sort of insoluble support medium. Separation is obtained since different molecules have different degree of interaction with ion exchanger due to difference in their charges, charge densities and distribution of charge on their surface.

Applications:

• A technique for separating two proteins differing by only one charged amino acid.
• It is capable of separating species with very minor differences in their properties.

The ion-exchange may be

• Anion exchanger or
• Cation exchanger.

(ii) Colorimetry:
Principle : It is based on the use of interaction of light energy with coloured solutions of certain molecules as when light passes through a coloured solution, some wavelengths are absorbed more than others. The amount of light absorbed is proportional to the intensity of colour and hence to the concentration of the compound.

Applications:

• Quantitative estimation i.e. concentration in solution.
• Detect and identification of biomolecules

(c) Inborn metabolic disorders are due to change in the gene, for the particular character. Some autosomal genes become recessive and are transmitted to the progeny.
Examples:

1. Albinism
2. Cystic fibrosis
3. Phenylketonuria
4. Sickle-cell anaemia
5. Alkaptonuria

Alkaptonuria: This was one of the first metabolic diseases described by Garrod in 1908. It is an inherited metabolic disorder produced due to deficiency of an oxidase enzyme requiredfor breakdown ofhomogentisic acid (also called alcapton, hence, alkaptonuria is also written as alcaptonuria). Lack of the enzyme is due to the absence of the normal form of gene that controls the synthesis of the enzyme. Hence, homogentisic acid then accumulates in the tissues and is also excreted in the urine. The most commonly affected tissues are cartilages, capsules of joints, ligaments and tendons. The urine of these patients if allowed to stand for some hours in air, turns black due to oxidation of homogentisic acid.

Albinism: It is caused by the absence of the enzyme tyrosinase which is essential for the synthesis of the pigment from dihydroxyphenylalanine. The gene for albinism (a) does not produce the enzyme tyrosinase but its normal allele (A) does. Thus, only homozygous individual (aa) is affected by this disease. Albinos (individuals with albinism) lack dark pigment melanin in the skin, hair and iris. Although albinos have poor vision yet they lead normal life.

Question 6.
(a) Discuss the significance of each of the following techniques used in cell culture technology: [4]
(i) Androgenesis and gynogenesis
(ii) In-vitro pollination
(b) Enumerate any two post transcriptional changes in the mRNA to produce a completely mature mRNA. [4]
(c) State any four achievements of the Human Genome Project. [2]
Answer:
(a) (i) Androgenesis involves production of haploid plants by development of an egg cell
containing male nucleus. The female nucleus is eliminated before fertilization. Gynogenesis includes production of haploid plants by the development of an unfertilized egg cells because of delayed pollination (through use of abortive pollen pre-exposed to ionizing radiations or using an alien pollen). It is found in inter-specific crosses of potato.

(ii) In vitro pollination : A laboratory procedure, in which pollination and fertilisation occur under in vitro condition, offer an opportunity for producing hybrid embryoids among plants that cannot be crossed by conventional method of plant breeding. To overcome interspecific and intergeneric incompatibility cases, the style or part of it can be excised and pollen grains are either placed on the cut surface of ovary or transferred through a hole in the wall of ovary. This technique is called intra-ovarian pollination. It is applied successfully in Papaver somniferum, Argemone mexicana etc. It also involves direct pollination of cultured ovules or excised ovules together with placenta.

(b) Post-transcriptional changes : Modification at 5′ end of primary RNA (Heterogenous RNA or hnRNA) transcript is called capping. During capping, an extra guanosine residue in first added to the terminal nucleoside triphosphate of the primary RNA transcript. Guanosine residue is further modified by addition of methyl groups. Capping prevents the 5′ end of TORNA from being digested by exonucleases. It aids in the transport of m-RNA out of the nucleus and plays an important role in the m-RNA translation.

Addition of a string of adenosine residues, forming a poly (A) tail at 3′ end of the TO-RNA.

The process of removal of those parts of primary transcript, corresponding to introns and joining together of exons, is known as splicing.

(c) Achievement of Human Genome Project:

• Human genome is largely consisting of – 3 × 109 base pairs a lot of repeated sequences.
• 3 billion base pairs were translated into biologically meaningful information by using computers and it led to a new field of bioinformatics.
• Human genome sequencing will enable a new approach to biological research.
• Understanding of function of biological systems increases in public and private sectors due to knowledge from DNA sequences.
• More than 1200 genes are associated with common human disorders such as Cardio- . Vascular diseases, diabetes, (endocrine disease), Alzheimer disease (neurological disorder).
• Efforts for health care are being made to design drugs, genetically modified diets, nutraceuticals etc.

Question 7.
(a) Explain the method used for the construction of a genomic DNA library. Also state how a genomic DNA library differs from a cDNA library. [4]
(b) Discuss any four vector-less methods of transfer of foreign DNA into the host cells. [4]
(c) Define glycosidic bond and peptide bond. [2]
Answer:
(a) DNA Library: DNA library is a collection of DNA fragments of one organism, each carries by a plasmid or virus and cloned in an appropriate host. A DNA probe is used to locate specific DNA sequence in the library. A collection representing the entire genome is called genomic (DNA) library. An assortment of DNA copies of messenger RNA produced by a cell is known as a complimentary DNA (cDNA) library.

Construction of Genomic Library: The process of subdividing genomic DNA into clonable elements and inserting them into host cells is called creating a library.

A complete library, by definition, contains the entire genomic DNA of the source organism and is called as genomic library. A genomic library’ is a set of cloned fragments of genomic DNA.

The process of creating a genomic library includes four steps:

1. In the first step the high molecular weight genomic DNA is separated and subjected to restriction enzy me digestion by using two compatible restriction enzy mes.
2. In the second step, the fragments are then fractionated or separated by using agarose gel electrophoresis to obtain fragments of required size.
3. These fragments are then subjected to alkaline phosphatase treatment to remove the phosphate. In the third step, the dephosphorylated insert is ligated into vector which could be a plasmid, phage or cosmid, depending upon the interest of the researcher.
4. In the last step, the recombinant vector is introduced into the host by electroporation and amplified in host.

In principle, all the DNA from the source organism is inserted into the host, but this is not fully possible as some DNA sequences escape the cloning procedure. Genomic library is a source of genes and DNA sequences. A genomic library is a set of cloned fragments of genomic DNA. Prior information about the genome is not required for library construction for most organisms. In principle, the genomic DNA, after the isolation, is subjected to RE enzyme for digestion to generate inserts.

cDNA libraries V/s Genomic libraries:

• Genomic library is a mixture of fragments of genomic DNA while cDNA obtained from ZM-RNA may cloned to give rise to a cDNA library. Genomic library contains DNA fragments that represent genes as well as those that are not genes. In contrast cDNA library contains only those genes that are expressed in the concerned tissue/organism. In both cases, a mixture of fragments is used for cloning to establish the library.
• Use of cDNA is absolutely essential when the expression of an eukaryotic gene is required in a prokaryote.
• Eukaryotic cDNAs are free from intron sequences.
• As a result of the above, they are smaller in size than the corresponding genes, i.e.. the genes that encoded them.
• A comparison of the cDNA sequence with the corresponding genome sequence permits the delineation of intron/exon boundaries.
• The contents of cDNA libraries from a single organism will vary widely depending on the developmental stage and the cell type used for preparation of the library. In contrast the genomic libraries w ill remain essentially the same irrespective of the developmental stage and the cell type used.
• A cDNA library will be enriched for abundant mRNAs. but may contain only a few or no clones representing rare mRNAs.

(b) Transfection: Transfection is the transfer of foreign DNA into cultured host cells mediated through chemicals. The charged chemical substances such as cationic liposomes, calcium phosphate of DEAE dextran are taken and mixed with DNA molecules. The recipient host cells are overtaxed by this mixture. Consequently the foreign DNA is taken up by the host cells.

Electroporation (Electric Field-mediated Membrane Permeation): In electroporation an electric current at high voltage (about 350 V) is applied in a solution containing foreign DNA and fragile host cells. This creates transient microscopic pores in cell membrane of naked protoplasts. Consequently foreign DNA enters into the protoplast through these pores. The transformed protoplasts are cultured in vitro which regenerate respective cell walls.

Microinjection : In this technique foreign DNA is directly and forcibly injected into the nucleus of animal and plant cells through a glass micropipette containing very fine tip of about 0.5 mm diameter. It resembles with injection needle. In 1982, for the first time Rubin and Spradling introduced Drosophila gene into P-element and microinjected into embryo.

Particle Bombardment Gun (Biolistics): This technique was developed by Stanford in 1987. In this method macroscopic gold or tungsten particles are coated with desired DNA. A plastic micro-carrier containing DNA coated gold/tungsten particles is placed near rupture disc. The particles are bombarded onto target cells by the bombardment apparatus. Consequently foreign DNA is forcibly delivered into the host cells

(c) Glycosidic bond (- O -) is the bond formed between the OH group attached to an anomeric carbon atom of a monosaccharide can easily dehydrate with an -OH group attached to another monosaccharide leading to the formation of a disaccharide.

Peptide bond (-NHCO-) is the bond formed between the carboxylic group (COOH) of one amino acid w ith the amino group (NH2) of another amino acid, with elimination of w ater to form a dipeptide.

Question 8.
(a) Explain the secondary and the quaternary structure of proteins. Mention any two important functions of proteins. [4]
(b) Discuss the method used for DN A sequencing by A utomated DNA sequencing technique. [4]
(c) Give any one example each of in-situ and ex-situ conservation. [2]
Answer:
(a) Secondary Structure (2° structure) of Proteins: It is development of new steric relationships amongst the amino acids for protecting their peptide bonds through formation of intrapolypeptide and interpolypeptide hydrogen bonds. Secondary’ structure is of three types – α-helix β-pleated and collagen helix. The prefixes α and β signify the first and second types of secondary structure discovered by Pauling and Corey (1951).

(i) α-Helix: The polypeptide chain is spirally coiled, generally in a clockwise or right-handed

fashion (Fig.). There are 3.6 amino acid residues per turn of the spiral. The spiral is stabilized by straight hydrogen bonds between imide group (-NH-) of one amino acid and carbonyl group (—CO—) of fourth amino acid residue. In this way all the imide and carbonyl groups become hydrogen bonded. R-groups occur towards the outer side of a-helix. a-helix is the final structure in certain fibrous proteins, e.g., keratin (hair, nail, horn), epidermis (skin).

β-Pleated Sheets : Two or more polypeptide chains come together and form a sheet. Condensation is little. How ever, tw isting does occur. The same polypeptide may fold over itself to form two strands for p-pleating. Adjacent polypeptide chains may occur in parallel (e.g., p-keratin) or anti-parallel (e.g., silk fibrin). Straight hydrogen bonds occur between imide (-NH-) group of one polypeptide and carbonyl (-CO-) group of adjacent polypeptide. Cross-linkages help in stabilization of β-pleated sheets.

Collagen Helix (Fig.) : Collagen has a large amount of glycine (25%) and proline (and hydroxyproline, 25%). It cannot form a-helix due to them. Three of its polypeptide each having about 1000 amino acid residues, come together with each forming an extended left-handed helix. They run parallel, form a right-handed super-helix that is stabilised by hydrogen bonds amongst the three. The triple helix of collagen is often called tropo- collagen. Its one end is stabilised by -S-S- linkages amongst the three chains. Collagen occurs in those tissues where extensibility is limited, e.g., connective tissue, tendons, bones.

Quaternary Structure (4° Structure) of proteins : It is the last or fourth level of protein organisation found in only oligomeric proteins or multimers. The multimeric proteins are formed of two to several polypeptides. The monomers or polypeptide subunits are also called protomers. Protomers may be similar, e.g., two similar polypeptides in enzyme phosphorylase a. It is known as homogeneous quaternary structure. An oligomeric protein having dissimilar subunits shows heterogeneous quaternary structure, e.g., tetrameric haemoglobin with two a (141 amino acids each) and two β (146 amino acids each) polypeptide chains.

Functions of proteins:

• Major group of proteins are enzymes-biocatalyst.
• Myoglobin, a protein found in muscle store oxygen.
• Haemoglobin in RBC’s transports gases in and out of lungs.
• Proteins as structural element; as hairkeratins and bio-membranes.

(b) Automatic DNA Sequencing : In this new method a different fluorescent dye is tagged to the ddNTPs. Using this technique a DNA sequence containing thousands of nucleotides can be determined in a few hours. Each dideoxynucleotide is linked with a fluorescent dye that imparts different colours to all the fragments terminating in that nucleotide. All four labelled ddNTPs are added to a single capillary tube. It is a refinement of gel electrophoresis which separates fastly. DNA fragments of different colours are separated by their respective size in a single electrophoretic gel. A current is applied to the gel. The negatively charged DNA strands migrate through the pores of gel towards the positive end.

The small sized DNA fragments migrate faster and vica versa. All fragments of a given length migrate in a single peak. The DNA fragments are illuminated with a laser beam. Then the fluorescent dyes are excited and emit light of specific wavelengths which is recorded by a special ‘recorder’. The DNA sequences are read by determining the sequence of the colours emitted from specific peaks as they pass the detector. This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by the computer (Fig.). Colour of each dye represents the different nucleotides. Computer converts the data of emitted light in the nucleotide sequences.

(c) In situ conservation refers to ‘on-site’ conservation of plants and animals as such. It is a process of protecting plants or animals in its natural habitat by protecting or cleaning up the habitat, or by defending the species from the predators, e.g., National Park, Sanctuaries and Biosphere Reserve.

Ex situ conservation is ‘off-site conservation’ by protecting an endangered species of plant or animal by removing part of population from a threatened habitat and placing it in a new location such as wild area, zoos or botanical gardens, in vitro gene bank, germplasm-banks, in vivo gene bank.

Question 9.
(a) What is meant by the term genomics ? Mention the difference between structural genomics and functional genomics. [4]
(b) How do the following databases contribute towards managing biological data : [4]
(i) GDB and MGD
(ii) PDB and PIR
(c) Name any two organisms whose genomes have been completely sequenced. [2]
Answer:
(a) Genomics is a scientific discipline of mapping, sequencing and analysing the genome-the complete set of chromosomal and extra-chromosomal genes of an organism. Structural genomics deals with DNA sequencing, sequence assembly, construction of genetic, physical or sequence maps of high resolution of the organism.

Functional Genomics deals with reconstructing genome sequences and to find out the functions they do. It provides novel information about the genome. It helps in understanding of genes and functions of proteins and protein interactions.

(b) (i) GDB (Genome Database): It is the official central repository for genome mapping data created by Human Genome Project. Its central node is located at the hospital for sick children. GDB holds a vast quantity of data submitted by hundreds of investigators. The GDB has many useful genome resource web-links on its resource page.

MGD (Mouse Genome Database) is the primary public mouse genomic catalogue resource. The MGD includes information on mouse genetic markers and nomenclature, molecular segments, phenotypes, comparative mapping data, graphical display of linkage, cytogenetic and physical maps.

(ii) PDB (Protein Data Bank) : This database has the sequence of those proteins, nucleic acid, whose 3-D structures are known by crystallography or NMR spectroscopy.
Source : NCBI-USA; EBI, UK.

PIR (Protein Information Resource): It is an integrated public bioinformatics resource to support genomic, proteomic and system biology, research and scientific studies.

(c) Organisms with completely sequenced genome : Phage λ , HIV, E.coli, Heliobacterpylori, Saccharomyces cerevisiae (yeast), Drosophila melanogaster (fruitfly). (any one organism)

ISC Class 12 Biotechnology Previous Year Question Papers

ISC Biotechnology Previous Year Question Paper 2017 Solved for Class 12

Maximum Marks: 80
Time allowed: Three hours

• Candidates are allowed additional 15 minutes for only reading the paper. They must NOT start writing during this time.
• Answer Question 1 (Compulsory) from Part I and five questions from Part II, choosing two questions from Section A, two questions from Section B and one question from either Section A or Section B.
• The intended marks for questions or parts of questions are given in brackets [ ].
• Transactions should be recorded in the answer book.
• All calculations should be shown clearly.
• All working, including rough work, should be done on the same page as, and adjacent to the rest of the answer.

Part -1 (20 Marks)
(Answer all questions)

Question 1.
(a) Mention any one significant difference between each of the following : [5]
(i) Reducing sugar and non-reducing sugar.
(ii) Triploids and haploids.
(iii) Lac operon and Trp operon
(iv) Blunt end and sticky end
(v) Spectroscopy and colorimetry

(b) Answer the following questions : [5]
(i) Who developed the microbe called super bug, which was designed to degrade spilled oil ?
(ii) Name any two growth regulators used in a culture medium.
(iii) What is an apoenzyme ?
(iv) How is the disease albinism caused ?
(v) State any one limitation of gynogenesis.

(c) Write the full form of each of the following : [5]
(i) AFLP
(ii) SSBs
(iii) BAC
(iv) CIMAP
(v) PAGE

(d) Explain briefly : [5]
(i) Polyadenylation
(ii) Lock and key model of enzyme action
(iii) Edible vaccine
(iv) Vascular differentiation
(v) Seedless crops
Answer:
(a) (i) Reducing sugar possess a free aldehyde (–CHO) and ketone (–C = 0) group while non-reducing have no free aldehyde and ketone group.

(ii) Plants produced from pollen grains or egg cells of ovules are called haploid plants. Plants produced from endosperm are called triploid plants.

(iii) Lac-operon is under negative gene control.
Trp-operon is under positive gene control.

(iv) Blunt ends : Some restriction enzymes cut both the strands of a DNA molecule at the same site so that the resulting termini or ends have blunt or flush ends in which the two strands end at the same point.

Sticky ends : Most of the restriction enzymes produce staggered cuts in which the two strands of DNA double helix are cleaved at different locations producing two protuding end (3′ or 5′) called cohesive or sticky ends. They readily pair with each other under annealing conditions. When fragments generated by a single restriction enzyme from different DNA are mixed they join together due to their sticky’ ends.

(v) Spectroscopy is the study of the interaction between matter and electromagnetic radiation. Colorimetry involves the estimation of the concentration of a colored substance in a solution by comparing the intensity’ or depth of its color with that of a solution having known concentration of the substance while passing visible light of the same intensity through both the solutions.

(b) (i) Ananda Mohan Chakrabarty engineered a new species of Pseudomonas bacteria in 1971. This is the oil spill disester bacteria (super bug).

(ii) Growth regulator hormones : These are Auxin and Cytokinins. Auxins (Indole acetic acid, 1-napthaleneacetic acid), Cytokinins (6-benzylaminopurine, zeatin).

(iii) Apoenzyme: Protein that forms an active enzyme system by combination with a coenzyme and determines the specificity’ of this system for a substrate.

(iv) Albinism: It results from inheritance of recessive alleles due to non-conversion of tyrosine into melanin.

(v) Limitation of Gynogenesis:

• Frequency’ of responding ovules is quite low i.e., 1-5 %
• Successful only is relatively small number of species.

(c) (i) AFLP: Amplified Fragment Length Polymorphism.

(ii) SSB’s : Single-Strand Breaks or Single-Strand DNA Binding Proteins.

(iii) BAC : Bacterial Artificial Chromosome.

(iv) CIMAP: Central Institute of Medicinal and Aromatic Plants.

(v) PAGE : Poly Acrylamide Gel Electrophoresis

(d) (i) Polyadenylation: It is the addition of a poly (A) tail to a messenger RNA. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation.

(ii) Lock and key mechanism : This model was proposed by Emil Fisher in 1898. It is also called the template model. According to this model the union of the substrate and the enzyme takes place at the active site, more or less in a manner in which a key fits in a lock and results in the formation of an enzyme substrate complex. As the two molecules are involved, this hypothesis is also known as the concept of inter-molecular fit. The ES complex is highly unstable and almost immediately this complex breaks to produce the end product of the reaction and regenerate the free enzyme. The ES complex results in the release of energy.

(iii) Edible vaccine are the antigenic proteins that induce B-cells to secrete antibodies. Transgenic crop plants can be constructed which produce vaccine to be eaten i.e., edible vaccine on a large scale at low cost. In 1990, first report of production of edible vaccine in tobacco was establish. There are many advantages associated with edible vaccine such as no problem of storage, easy delivery’, low cost of production. These provide similar effects as the recombination vaccines.

(iv) Vascular differentiation : In vascular differentiation, first identify and clone transcripts that are produced in differentiating vascular tissues, then approach gene function by assessing phenotype in plants with loss of function, created by insertion mutagenesis of RNA interference.

(v) A seedless fruit is a fruit developed to possess no mature seeds. As consumption of seedless crops e.g., fruit is generally easier and more convenient, they are considered commercially valuable. Most commercially produced seedless fruits have been developed from plants whose fruits normally contain numerous relatively large hard seeds distributed throughout the flesh of the fruit.

Seedless fruits can develop in one of two ways : either the fruit develops with fertilization (parthenocarpy), or pollination triggers fruit development, but the ovules or embryos abort without producing mature seeds (stenospermocarpy). Seedless banana and watermelon fruits are produced on triploid plants, whose three sets of chromosomes make it very unlikely for meiosis to produce fertile gametes.

One disadvantage of most seedless crops is a significant reduction in the amount of genetic diversity in the species. As genetically identical clones, a pest or disease that affects one individual is likely capable of affecting each of its clones.

Part – II (50 Marks)
(Answer any five questions)

Question 2.
(a) Briefly explain the structure of tRNA. Write its function in protein synthesis.
(b) With reference to lipids, explain its :
(i) Building blocks.
(ii) Any two chemical properties.
(c) What is DNA probe ?
Answer:
(a) Transfer RNA (tRNA) : It is also called soluble or sRNA. There are over 100 types of tRNA’s. Transfer RNA constitutes about 15% of the total RNA. tRNA is the smallest RNA with 70-85 nucleotide’s and sedimentation coefficient of 4S. The nitrogen bases of several of its nucleotide’s get modified e.g., pseudouridine (φ), dihydrouridine (DHU), inosine (I). This causes coiling of the otherwise single- stranded tRNA into L-shaped form (three dimensional, Klug, 1974) or clover-like form (two dimensional, Holley, 1965). About half of the nucleotides are based paired to produce paired stems. Five regions are unpaired of single-stranded —AA-binding site, T φ C loop, DHU loop, extra arm and anticodon loop,

(i) Anticodon. It is made-up of three nitrogen bases for recognizing and attaching to the codon of tRNA.
(ii) AA-Binding site. It lies at the 3′ end opposite to the anticodon and has CCA—OH group (5′ ends bears G). Amino acid or AA-binding site and anticodon are the two recognition sites of tRNA.
(iii) T φ C loop. It contains pseudouridine. The loop is the site for attaching to ribosomes.
(iv) DHU loop. The loop contains dihydrouridine. It is binding site for aminoacyl synthetase enzyme,
(v) Extra arm. It is a variable site arm or loop which lies between T φ C loop and anticodon. The exact role of extra arm is not known.

Functions :
(i) tRNA is adapter molecule which is meant for transferring amino acids to ribosomes for synthesis of polypeptides. There are different tRNAs for different amino acids. Some amino acids can be picked up by 2-6 tRNA’s. tRNA’s carry specific amino acids at particular points during polypeptide synthesis as per codons of wRNA. Codons are recognised by anticodons of tRNA’s. Specific amino acids are recognized by particular activating or aminoacyl synthetase enzymes.

(ii) They hold peptidyl chains over the mRNA’s. The initiator tRNA has the dual function of initiation of protein synthesis as well as bringing in of the first amino acids. There is, however, no tRNA for stop signals.

(b) (i) The building blocks of lipids are the long chain hydrocarbons, glycerol and cholesterol.
They are special group of lipids. Building blocks of lipids show various types of reactivities of ester linkage and the degree of unsaturation of hydrocarbon chain. Simple fatty acids, Sphingosine, Glycerol and Cholesterol these are the building blocks of lipids.

(ii) Chemical properties of Lipids :
Hydrolysis: On hydrolysis with alkali or lipolytic enzyme lipases, fats are broken down into their components fatty acids and glycerols.

Saponification : The hydrolysis of fats by alkali to produce glycerol and soaps (salts of fatty acids) is known as saponification.

(c) DNA Probe : It is a solution of radioactive, single-stranded DNA or oligodeoxy nucleotides (a DNA segment of few to several nucleotide’s). The name probe signifies the fact that this DNA molecule is used to detect and identify the DNA fragment in the gel membrane that has a sequence complementary to the probe. The probe hybridizes with the complementary DNA on the membrane to the greater extent with a low non-specific binding on the membrane. This step is known as hybridization reaction.

Question 3.
(a) Explain the process involved in the transcription of DNA to mRNA. [4]
(b) What are stem cells ? Explain the various types of stem cells. [4]
(c) Name any two chemicals used to determine the amino acid sequence in protein. [2]
Answer:
(a) Transcription is the process of creating a messenger RNA strand from DNA, performed by the enzyme RNA polymerase. Transcription always occurs in a 5’→ 3′ direction, with polymerase moving 3’→ 5′ along the DNA strand.

Transcription Initiation : There are three steps in transcription :
(1) Initiation: RNA synthesis begins after the RNA polymerase attaches to the DNA and unwinds it. RNA synthesis will always occur on the template strand.

(2) Elongation: RNA polymerase unwinds the DNA double helix and moves downstream and elongates the RNA transcript by adding ribonucleotides in a ‘5 →3′ direction. Each ribonucleotide is added to the growing mRNA strand using the base pairing rules (A binds with T, G binds with C). For each C encountered on the DNA strand a G is inserted in the RNA, for each Q a C and for each T, an A is inserted. Since there is no T in RNA, U is inserted whenever an A is encountered. After RNA polymerase has passed, the DNA restores its double stranded structure.

(3) Termination: When the mRNA is complete, the mRNA is released and the RNA polymerase releases from the DNA.
Two post transcriptional changes that occur in the mRNA formed are :
RNA transcripts Eukaryotas are modified or processed, before leaving the nucleus to produce functional mRNA. It is processed in two ways :
(1) 5′ capping: Capping of the pre-mRNA involves the addition of 7-methylguanosine to the 5′ end.,

(2) 3′ poly adenylation : The pre-mRNA processing at the 3′ end of the RNA molecule involves cleavage of its 3′ end and then the addition of about 200 adenine residues to form a poly (A) tail. The cleavage and adenylation reaction occur if a polyadenylation signal sequence (5′ – AAU AAA-3′) is located near the 3′ end of the pre-mRNA molecule, followed by another sequence, which is usually (5′-CCA-3’)

(b) Stem cells are the cell which are capable to divided and renew and also produce progeny. These can differentiate into a variety of different cells types, e.g., tissues continuously renew themselves throughout the life. The stem cells have two properties which increases their importance.
(i) They have potential to form more differentiated cells and
(ii) these are self-renewing because each division of a stem cells creates at least one stem cell.

Various types of stem cells : On the basis of development potential, these are divided into following levels:

• Totipotent Cells : which give rise to entire organism.
• Pluripotent Cells : w hich form totipotent cells and also give rise to most but not all of cell types that are necessary for foetal development.
• Multipotent Cells : cells which are formed after further differentiation of pluripotent cells. These can give rise to a limited number of cells types.
• Unipotent Cells : are also formed from further differentiation of multipotent cells. These give rise to a single cell type.

(c) There are many chemicals which are used to determine the amino acid sequence in protein. Sanger’s reagent.(1-fluoro-2, 4-dinitrobenzene) and dansyl derivatives such as dansyl chloride are used for amino acid sequencing.

Question 4.
(a) Explain the following methods of selection of recombinant cells: [4]
(i) Insertional inactivation.
(ii) Blue white colony.
(b) Enumerate the steps involved in regenerating a plant from a single cell [4]
(c) What is wobble effect ? [2]
Answer:
(a) (i) Insertional inactivation: Harder problem to solve is to determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain self- ligated vector molecules. Insertional inactivation is the inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. Recombinants can, therefore, be identified because the characteristic coded by the inactivated gene is no longer visible.

pBR322 contains genes which code for ampicillin resistance and tetracycline resistance. BamHl cuts in the middle of the gene which codes for tetracycline resistance. If a gene is inserted here, the plasmid loses it ability to code for tetracycline resistance. Thus, the plasmid containing the recombinant gene is resistant to ampicillin but sensitive to tetracycline. To screen, we use replica plates.

(ii) The blue-white screen is a molecular technique that allows for the detection of successful ligation’s in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell (bacteria). In this method, a reporter gene lac Z is inserted in the vector. The lac Z encodes for the enzyme β- galactosidase which breaks a synthetic substrate X-gal (5-bromo-4-chloro-indolyl, (β-D-galacto-pyranoside) into insoluble blue colored product. The competent cells are grown in the presence of X-gal. If the ligation was successful, the bacterial colony will be white because p- galactosidase is not synthesized due to the inactivation of lac Z; if not, the colony will be blue. This technique allows for the quick and easy detection of successful ligation, without the need to individually test each colony. An example of such a vector is the artificially reconstructed plasmid pUC 19.

(b) Basic technique of Plant Tissue Culture : The basic technique of plant tissue culture involves the following steps :
Preparation and Sterilization of Suitable Nutrient Medium : Suitable nutrient medium as per objective of culture is prepared and transferred into suitable containers. Culture medium is rich in sucrose, minerals, vitamins, and hormones. Yeast extract, coconut milk, are also added. The culture is completely sterilized in an autoclave.

Selection of explants : Selection of explants such as shoot tip should be done.

Sterilisation of explants : Surface sterilisation of the explants by disinfectants (e.g sodium hypochlorite or mercuric chloride) and then washing the explants with sterile distilled water is essential.

Inoculation : Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilised by autoclaving to avoid microbial contamination) in culture vessels under sterile conditions is done.

Incubations : Growing the culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e., artificial light; 16 hours of photoperiod), temperature (- 26°C) and relative humidity (50-60%) is required.

Regeneration: An unorganized mass of cells developing from explants is called callus. The callus gives rise to embryoids which can develop into w hole plant if the medium is provided with proper concentration of hormones. This property of developing every somatic cell into a full fledged plant is called totipotency. Regeneration of plants from cultured plant tissues is carried out.

Hardening: Hardening is gradual exposure of plantlets to an environmental conditions.

Plantlet transfer: After hardening, plantlets are transferred to the greenhouse or field conditions following acclimatization (hardening) of regenerated plants.

(c) Wobble effect:
The wobble effect is an effect caused by the redundancy found in the genetic code. Each amino acid is coded by a 3 nucleotide sequence on the wRNA. The triplets are referred to as codons. Though there are only 21 amino acids, 61 of the 64 possible codons found on the mRNA code for amino acids (the other 4 codons signal translation stop and start points). Thus, each amino acid can be coded by more than one codon. For any amino acid, the first 2 nucleotide’s in the codon are always identical. It is the 3rd nucleotide that can change. This is where the wobble comes in.

Question 5.
(a) Discuss the working of PCR technique in detail.
(b) Explain the principle and any two applications of each of the following biochemical techniques:
(i) Iso-electric focusing.
(ii) Centrifugation.
(c) Where do we find the following carbohydrates:
(i) Glycogen
(ii) Chitin
Answer:
(a) Working mechanism of PCR: The action of PCR involves several cycles. However, there are three steps in one amplification cycles, e.g., denaturation (melting), annealing and polymerisation.

Denaturation : The two strands of DNA are separated by applying a high temperature (95°C). After separation each strand acts as template for DNA synthesis.

Primer annealing : Since nucleotide sequence of each oligonucleotide primer is complementary to 3′ end of single stranded template, the primers anneals (hy bridizes) the each template. Annealing is done at low temperature based on length and sequence of the primers. The annealing temperature varies, but the too low temperature favours mispairing. The annealing temperature (°C) can be calculated using the formula T = 2 (AT) + 4(G + C)

Extension (Polymerization): It is the final step of amplification cy cle. In the presence of Mg⁺⁺ and dNTP’s (deoxynucleotide triphosphates, e.g., dATP, dGTP, dCTP, dTTP), Taq DNA, polymerase (at 70°C for 60-90 seconds) synthesis through onward extension of primer in 5′ -» 3′ direction on single DNA template. The concentration of Mg⁺⁺ is maintained between 1 and 4 mM. Thus, in the first step, the target DNA is copied from the primer sites for various distances until the start of second cycle.

The second cycle starts and doubles the DNA molecules synthesized in the first cycle. The second cycle is started with heating of double stranded DNA to result in single DNA. Each single stranded DNA again acts as template;. e., DNA molecules polymerized in first cycle act as template in second cycle. Following the above events all the single stranded DNA molecules of second cycle are converted into the double stranded DNA. The third and onward cycles are repeated in the same ways to get more DNA products. Always after n number of cycles, 2n molecules of DNA are generated using single stranded DNA as template.

(b) (i) Isoelectric focusing (IEF), also known as electrofocusing. is a technique for separating different molecules by their electric charge differences. It is a type of zone electrophoresis usually performed on proteins in a gel.

(ii) Centrifugation: On the basis of physical properties, cells are separated. For example, the larger and smaller cells are separated by centrifugation. Different components of homogenate are separated on the basis of their shape, size and density. For the separation of cell components high speed centrifuges are used .e.g., zonal centrifuge, density gradient centrifuge.

(c) (i) Glycogen is also known as animal starch and it is the reserve of carbohydrates in animals. It occurs in algae, fungi and yeasts. Glycogen is dextro-rotatory and its hydrolysis yield D- glucose.

(ii) Chitin : It is hard,tough substance that occurs widely in nature particulary in the shells of arthropods such as crab, insects and spider. The walls of hyphae are composed of slightly different chitin. Chemically, chitin is a polysaccharide drived from glucose.

Question 6.
(a) Describe the procedure of sequencing of DNA by Sanger’s method. [4]
(b) Explain any two physical and any two chemical methods used to synchronize suspension cultures. [4]
(c) Name any two industrial enzymes and give their uses. [2]
Answer:
(a) DNA sequencing : It is the determination of the precise sequence of nucleotide’s in a sample of DNA.

Sanger dideoxy method: The most popular method for DNA sequencing is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded the (1980) Nobel prize in chemistry).

Procedure : The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with

a mixture of all four normal (deoxy) nucleotides in ample quantities

• dATP
• r/GTP
• c/CTP
• dT TP

a mixture of all four dideoxynucleotides, each present in limiting quantities and each labelled with a ” tag that fluoresces a different color:

• ddATP
• ddGTP
• ddc TP
• ddlTP

DNA polymerase I

Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines).

If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.

(b) Cells in. suspension cultures vary greatly in size, shape of DNA and nuclear content. Moreover, the cell cycle time varies considerably within individual cells. Therefore, cell cultures are mostly asynchronous. This variation complicates studies of biochemical, genetic physiological and other aspects of cell metabolism. A synchronous culture is one in which the majority of cells proceed through each cell cycle phase (G, S,G₂ and M) simultaneously.

(A) Physical Methods:
Selection by Volume: Synchronization may be achieved on the basis of selecting the size of cell aggregates present even in the finest possible suspension cultures. Cell fractionation is employed for selection.

Temperature Shock: Low temperature shocks combined with nutrient starvation are reported to induce — synchronization of suspension culture.

(B) Chemical Methods:
Starvation : The principle of starvation is based on depriving suspension cultures of an essential growth compound leading to a stationary growth phase. Resupplying the missing compounds is expected to induce resumption of cell growth synchronously. Growth hormone starvation is also reported to induce synchronization of cell cultures.

Inhibition : Synchronization is achieved by temporarily blocking the progression of events in the cell cycle and accumulating cells in a specific stage using a biochemical inhibitor. On release the block cells with synchronously enter the next stage. Inhibitors of DNA synthesis (5-aminourail, 5-fluorodexypunne. hydroxyurea or excess thymidine) in cell cultures accumulate cells at the G₁/S boundary.

(c) Tw o industrial enzymes with uses :
Amylases (from fungi and plants) : Production of sugars from starch, such as in making high fructose corns syrup.
Rennin (derived from the stomach of young ruminant animals): Manufacture cheese used to hydrolyze protein.

Question 7.
(a) Briefly explain the essential features of a vector. [4]
(b) What is the principle of cryopreservation? Mention the steps of cryopreservation. [4]
(c) What is the importance of pH and solidifying agents in cell cultures ? [2]
Answer:
(a) Vectors are the DNA molecules that serve as vehicle to carry a foreign DNA sequence into a host.
Features:
A vector should have the following features :

• origin for replication (ori)
• have a selectable marker such as antibiotic resistance
• should be small in size.
• It must contain at least a unique recognition site for restriction enzyme.

Examples are : Plasmids, Viral DNA, Cosmids, YAC vector, BAC vector, etc.

(b) Cryopreservation involves cooling and storing cells in a frozen state at an extremely low temperature that allows them a higher chances of surviving when thawed. Orthodox seeds have a natural dormancy feature, which allows for their long term storage with little damage to DNA, provided they are kept in a cool, dry environment.

Steps of cryopreservation : Following are the steps of cryopreservation :
Freezing: Freezing generally requires a low molarity of cryoprotectants which are reletively less toxic for a wide range of plant cells. This can be used on a large number of samples which are processed simultaneously. In this technique we can use computerized freezer.

Vitrification : This step does not require an expensive controlled-freezing system. This is more suitable for plant cells which are sensitive to chilling.

(c) Importance of solidifying agents and pH in cell cultures : For preparing a cell culture or media we should always follow the criteria. Every nutrient and condition are important. Depending on the type of the plant cell or tissue used for culture, the composition of nutrient media vary. A cell culture generally require these constituents : Inorganic nutrients, carbon and energy sources, organic supplements, growth regulators, solidifying agents and pH.

Solidifying agents play important role to solidify the culture media properly. Mostly we use Agar for solidify the culture. Agar is a polysaccharide obtained from seaweed. Agar gel do not react with constituents of media and it does not digested by the plant enzymes.

pH affects the uptake of ions. Always optimum pH of the medium should be maintained before sterilization of the medium. pH between 5.0 to 6.0 is an optimum pH which is required for growth and development of cultured tissues.

Question 8.
(a) Explain how DNA technology has been used to create the following: [4]
(i) Tomatoes with delayed ripening
(ii) Bt crops
(iii) Virus free crops
(iv) Biodegradable plastic.
(b) List the functions of the following bioinformatics tools: [4]
(i) GENSCAN
(ii) ENTREZ
(iii) FASTA
(iv) PIR
(c) Name any two media used in plant tissue culture. [2]
Answer:
(a) (i) Flaw Save tomato is a plant having delayed fruit ripening. This was genetically modified by introducing the genes from a fish to reduce the freezer damage of the tomatoes. This gives a long life to tomatoes.

(ii) Bt crops : Bt cotton and Bt brinjal, these are insect-resistant plants which are produced by introducing a cry gene of Bacillus thuringiensis that encode Cry protein.

(iii) Virus free crops : Transgenic papaya, peeper, tobacco and tomatoes; these are virus free plants. By using tissue culture method we can produce disease-resistant plants.

(iv) Biodegradable plastic : The biodegradable plastic is made from lactic acid which is produced at the time of bacterial fermentation of plant materials like discarded stalks of corn. In the process, molecules of lactic acid are chemically grouped to form the biodegradable plastic. In fact, the biodegradable plastic is a material which has most of the properties of plastic except the property of being non-biodegradable. The biodegradable plastic polyhydroxyalkanates eg., polyhydroxylbutyrate (PHB) are obtained commercially by fermentation with bacterium Alcaligenes eutrophus. The genetically engineered Arabidopsis plants produced polyhydroxylbutyrate (PHB) globules in their chloroplasts without effecting plant growth and development. The large scale of polyhydroxylbutyrate (PHB) can be extracted from leaves as well as from transgenic plants.

(b) (i) GENSCAN: Genscan is a notable example of eukaryotic ab initio gene finders. It is one of the best gene finding algorithms for sequence alignment and gene prediction.

(ii) ENTREZ : This is an integrated information database retrieval system of NCBI. It is mainly used for accessing literature, sequences (both proteins and nucleotides) and structure (3D).

(iii) FASTA: It is a widely used programme for database similarity search. It performs optimised search for local alignment using a substituion matrix. FASTA format is used in a variety of molecular biology software suits.

(iv) PIR : PIR is a non-redundant annotated protein sequence database and analytical tools.

(c) There are different types of culture media which are used in plant tissue culture. We use tham as root culture medium, organogenesis medium, cell and callus medium initially.
Following are the medias :
1. Ms medium
2. White medium
3. B5 medium
Ms medium is mostly used in the tissue culture.

Question 9.
(a) What are restriction enzymes? How do they work? What are the different types of restriction enzymes? [4]
(b) Define the term proteomics. Explain the various types of proteomics. [4]
(c) Differentiate between the following: [2]
(i) Local alignment and Global alignment.
(ii) FST and STS.
Answer:
(a) The restriction enzymes are called ‘molecular scissors’. Restriction enzymes are DNA-cutting enzymes present in bacteria. They are obtained from them for use in genetic engineering-rDN A technology. As sequence are cut within the DNA molecule, they are often called restriction endonucleases.

A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named Hae III that cuts DNA whenever it identifies the recognition sequence.

A cut is made between the adjacent G and C. Hae III cleaves both the strands of DNA at the . same base pairs producing “blunt” ends.
Examples : EcoRI, Hind III and Bam HI.

(b) Proteomics : It is the identification, analysis and large scale production of total protein components of an organism.
Types of Proteomics : Generally, proteomics have so many types but we will only discuss its following three types :

1. Expression proteomics
2. Structural proteomics
3. Functional proteomics

Expression proteomics is the quantitative study of protein expression between the samples which differ by some variables.

Structural proteomics generally deals with the study of the structure and nature of protein complexes which are present in a particular cell organelles.

Functional proteomics generally embraces all proteomics approaches related to devising its functions. In this we use proteomics methods for analysis of properties of molecular networks that are formed in a living cell. This is a complex process.

(b) Global vs local alignments: Illustration of global and local alignments demonstrating the ‘gappy’ quality of global alignments that can occur if sequences are insufficiently similar.

Global alignments, which attempt to align every residue in every sequence, are most useful when the sequences in the query set are similar and of roughly equal size (This does not mean global alignments cannot end in gaps). A general global alignment technique is the Needleman-Wunsch algorithm, which is based on dynamic programming.

Local alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar sequence motifs within their larger sequence context. The Smith-Waterman algorithm is a general local alignment method also based on dynamic programming. With sufficiently similar sequences, there is no difference between local and global alignments.

EST is known as Expressed Sequence Tag. It is a unique stretch of DNA within a coding region of a gene that is useful for identifying full length genes and serves as a landmark for mapping. EST is a sequence tagged site derived form cDNA. STS is a short segment of DNA which occurs but once in the genome and whose location and base sequence are known. STS’s are detectable by PCR.

ISC Class 12 Biotechnology Previous Year Question Papers

ISC Biotechnology Previous Year Question Paper 2018 Solved for Class 12

Maximum Marks: 80
Time allowed: Three hours

• Candidates are allowed additional 15 minutes for only reading the paper. They must NOT start writing during this time.
• Answer Question 1 (Compulsory) from Part I and five questions from Part II, choosing two questions from Section A, two questions from Section B and one question from either Section A or Section B.
• The intended marks for questions or parts of questions are given in brackets [ ].
• Transactions should be recorded in the answer book.
• All calculations should be shown clearly.
• All working, including rough work, should be done on the same page as, and adjacent to the rest of the answer.

Part-I (20 Marks)
(Answer all questions)

Question 1.
(a) Mention any one significant difference between each of the following : [5]
(i) Plasmids and cosmids
(ii) Nucleotide and Nucleoside
(iii) Lagging strand and leading strand
(iv) Multipotent cells and unipotent cells
(v) Microinjection and biolistic
(b) Answer the following questions : [5]
(i) Who coined the term vitamin ? Write the chemical name of vitamin D.
(ii) Why is amino acid said to be amphoteric ?
(iii) What is Bioremediation ?
(iv) What is a primer ?
(v) What are cryoprotectants ?
(c) Write the full form of each of the following : [5]
(i) NBPGR
(ii) ARS
(iii) RELP
(iv) HEPA
(v) SCP
(d) Explain briefly : [5]
(i) Gene splicing
(ii) Supramolecular assembly
(iii) Interferon
(iv) Gene scan
(v) Saponification
Answer:
(a) (i) Plasmids and Cosmids
Plasmids: These are the extra-chromosomal, independent, self replicating, circular double stranded DNA molecules naturally found in all bacteria and some fungi.

Cosmids : These are predominantly plasmids with a bacterialori, an antibiotic selection marker and a cloning site, but they carry one or more recently two ‘cos’ sites derived from bacteriophage lambda.

(ii) Nucleotide and Nucleoside:
Nucleotide:
Nucleotide is a condensation product of heterocyclic nitrogen base, a pentose sugar like ribose or deoxyribose, and a phosphate or polyphosphate group.

Nucleoside:
Nucleoside consisting of a pentose sugar, usually ribose or deoxyribose, and a nitrogen base purine or pyrimidine.

(iii)Lagging strand and Leading strand
Lagging strand:

1. Lagging strand is a replicated strand of DNA which is formed in short segments called Okazaki fragments. Its growth is discontinuous.
2. DNA-ligase is required for joining Okazaki fragments.

Leading strand:

1. It is a replicated strand of DNA which grows continuously without any gap.
2. It does not require DNA ligase for its growth.

(iv) Multipotent cells and Unipotent cells
Multipotent Cells:
Multi potent cells have the ability to differentiate into many of the various types of specialized cell types and can develop into any cell of a particular group or type,
e.g., Umbilical cord stem cell

Unipotent Cells:
Uni potent cells can undergo unlimited reproductive divisions, but can only differentiate into a single type of cell or tissue.
e.g., Skin cells.

(v)Microinjection and biolistic
Microinjection:
In this technique foreign DNA is directly and forcibly injected into the nucleus of animal and plant cells through a glass micropipette containing very fine tip of about 0.5 mm diameter. It resembles with injection needle.

Biolistic:
In this technique macroscopic gold or tungsten particles are coated with desired DNA. The particles are bombarded onto target cells by the bombardment apparatus. Consequently foreign DNA is forcibly delivered into the host cells.

(b) (i) The term Vitamin was coined by Funk.
Chemical name of Vitamin D: Cholecalciferol (D3), Ergocalciferol (D2)

(ii) Amino acids are amphoteric, which means they have acidic and basic tendencies. The carboxyl group is able to lose a proton and the amine group is able to accept a proton.
Or
In neutral amino acid solution, the (– COOH) loses a proton and the – NH₂ of the same molecule picks up the one. The resulting ion is dipolar charged but overall electrically neutral. Due to this reason amino acids are amphoteric in nature i.e., they donate or accept proton.

(iii) Bio-remediation : Nowadays, microbial preparations are used for degradation of these pollutants. The waste organic materials treated with selected potential strains of microorganisms are rendered into less or non-toxic forms. This process of remedying pollution is called bio-remediation.

(iv) Primer: A primer is a short strand of RNA that serves as a starting point for DNA synthesis. They are required for DNA replication because DNA polymerases can only add new nucleotide’s to an existing strand of DNA.

(v) Cryoprotectants: These are basically antifreeze that is added to the solutions in which the cells are being stored to protect them from membrane damage and ice crystal damage. Examples are DMSO, Polyethylene glycol.

(c) (i) NBPGR: National Bureau of Plant Genetic Resources.
(ii) ARS: Agricultural Research Service.
(iii) RFLP: Restriction Fragment Length Polymorphism
(iv) HEPA: High Efficiency Particulate Air Filter
(v) SCP: Single Cell Protein

(d) (i) Gene splicing: In molecular biology, splicing is a post transcriptional modification of an hnRNA, in which introns are removed and exons are joined.

(ii) Supra molecular assembly: It is based on weak bonds as supposed to covalent bonds. As mentioned weak bond, though harder to control have the advantage of possible reopening. Relying on weak bond in surface organisation, can lead to molecular assemblies and raise the probability of reorganization processes.

(iii) Interferon: They are natural anti-viral proteins produced by the cells of the system of most vertebrates in response to attack of foreign agents such as viruses, parasites and tumor cells. These belong to the large class of glycoprotein known as cytokine. Today interferon’s are used for the treatment of Hepatitis-C diseases.

(iv) Gene scan: It is a notable example of eukaryotic ab initio gene finders. Gene scan is one of the best gene finding algorithms for sequence alignment and gene prediction.

(v) Saponification : The hydrolysis of triacvlghcerol in the presence of sufficient sodium hydroxide is called saponification.

Part-II (50 Marks)
(Answer any five questions)

Question 2.
(a) Explain in detail, how Dolly, the sheep was created. [4]
(b) Mention any two chemical properties of each of the following: [4]
(i) Proteins
(ii) Carbohydrates
(c) What are Okazaki fragments ? How are they joined ? [2]
Answer:
(a) Dolly, was the first mammal to be cloned by Wilmut et. al (1977). Dolly was the end result oi a long research program founded by the British government at the Roslin Institute in Scotland. They used the technique of somatic cell nuclear transfer, where the cell nucleus from an adult udder cell is transferred into an unfertilized oocyte that has had its nucleus removed. The hybrid cell is then stimulated to divide by an electric shock, and the blastocyst that is eventually produced’is implanted in a surrogate mother. Dolly was the first clone produced from a cell taken from an adult animal.

The following steps are involved in creation of Dolly:
Isolate donor nucleus : Isolate the nucleus from a somatic (non-reproductive) cell of a udder of adult donor sheep. The nucleus contains all the complete genetic material of the organism. A very small needle and syringe (suction device) is used to penetrate through the cell membrane to hold the nucleus and remove it from the cell.

Get unfertilized eggs: Retrieve some unfertilized egg cells (reproductive) from a female sheep. Many eggs are needed since not all of them will survive the various steps of cloning.

Remove the egg’s nucleus : Remove the egg cell’s nucleus, which contains only one-half of the sheep’s genetic material.

Insert donor nucleus : Insert the nucleus, with all its complete genetic material, isolated from the donor sheep mammal in Step 1 into the cytoplasm of egg cell that has no nuclear material. The egg’s genetic material now contains all traits from the donor adult. This egg is genetically identical to the donor adult cells.

Transfer the egg into womb : Transfer the egg into a receptive female sheep’s womb. Those eggs that survive and implant will continue to develop into embryos. When the off spring is born, it is a clone (genetically identical) of the donor sheep. After cloning was successfully demonstrated through the production of Dolly, many other large mammals have been cloned, including horses and bulls.


(b) Chemical properties of proteins:

1. Proteins when hydrolyzed by acidic agents, like cone. HCl yield amino acids in the form of their hydrochlorides.
2. Sanger’s reaction : Proteins react with FDNB reagent to produce yellow coloured derivative, DNB amino acid.
3. Xanthoproteic test: On boiling proteins with cone. HNO₃, yellow colour develops due to presence of benzene ring.
4. Folin’s test: This is a specific test for tyrosine amino acid, where blue colour develops with phosphomolybdotungstic acid in alkaline solution due to presence of phenol group.

Chemical properties of carbohydrates:
Carbohydrates with free hemiacetals form (having anomeric carbon) are called reducing sugars such as glucose, fructose, maltose, etc., They can reduce alkaline solution of copper salts giving rise to yellow to red precipitate of cuprous oxides.

The reducing sugars give positive test for Fehling’s test, Benedict’s test and Barfoed s test. For the detection of sugars in blood/urine, these tests are carried out in a routine way in the pathology laboratory.

(c) Okazaki fragments are short, newly synthesized DNA fragments produced discontinuously in pieces during DNA replication. They are formed on the lagging template strand and are complementary to the lagging template strand. Okazaki fragments are joined together by DNA ligase enzyme.

Question 3.
(a) Describe the effect of each of the following factors on enzyme activity. [4]
(i) pH
(ii) Temperature
(iii) Enzyme concentration
(iv) Concentration of products
(b) With reference to suspension culture, explain the following : [4]
(i) A chemostat
(ii) A turbidostat
(c) What is genomics ? What are its different types ? [2]
Answer:
(a) (i) Optimum pH : Every enzyme has an optimum pH when it is most effective. A rise or fall in pH reduces enzyme activity 7 by changing the degree of ionization of its side chains. A change in pH may also start reverse reaction. Fumarase catalyses fumarate → malate at 6.2 pH and reverse at 7.5 pH. Most of the intracellular enzymes function near neutral pH with the exception of several digestive enzymes which work either in acidic range of pH or alkaline, e.g., 2.0 pH for pepsin, 8.5 for trypsin.

(ii) Temperature: Higher temperature generally causes more collisions among the molecules and therefore, increases the rate of a reaction. More collisions increase the likelihood that substrate will collide with the active site of the enzyme, thus increasing the rate of an enzyme-catalyzed reaction. Above a certain temperature, activity begins to decline because the enzyme begins to denature. The rate of chemical reactions, therefore, increases with temperature but then decreases as enzymes denature.

(iii) Enzyme Concentration: If there is insufficient enzyme present, the reaction will not proceed as fast as it otherwise would because all of the active sites are occupied with the reaction. Additional active sites could speed up the reaction. As the amount of enzyme is increased, the rate of reaction increases. This is so because when more enzyme molecules are present, more substrate molecules can be acted upon at the same time. This means that the total substrate molecules are broken down quickly. If there are more enzyme molecules than are needed, adding additional enzyme will not increase the rate. Reaction rate therefore increases as enzyme concentration increases but then its levels off.

(iv) Effect of Product Concentration: The accumulation of reaction products generally decreases the enzyme velocity. For certain enzymes, the products combine with the active site of enzyme and form a loose complex and thus, inhibit the enzyme activity. In the living system, this type of inhibition is generally prevented by a quick removal of products formed.

(b) (i) Chemostat : A type of cell culture; a component of medium is in a growth limiting concentration; fresh medium is added at regular intervals and equal volume of culture is withdrawn. But in a chemostat, a chosen nutrient is kept in a concentration so. that it is depleted very rapidly to become growth limiting, while other nutrients are still in concentrations higher than required. In such a situation, any addition of the growth- limiting nutrient is reflected in cell growth. Chemostats are ideal for the determination of effects of individual nutrients on cell growth and metabolism

(ii) Turbidostat : A type of suspension culture; when culture reaches a predetermined cell density, a volume of culture is replaced by fresh medium; works well at growth rates close to the maximum. A continuous culturing method where the turbidity of the culture is kept constant by manipulating the rate at which medium is fed. If the turbidity falls, the feed rate is lowered so that growth can restore the turbidity to its start point. If the turbidity rise • the feed rate is increased to dilute the turbidity back to its start point.

(c) Genomics: Genomics is an area within genetics that concerns the sequencing and analysis of an organism’s genome. The genome is the entire DNA content that is present within and cell of an organism. Experts in genomics strive to determine complete DNA sequences and perform genetic mapping to help understand disease.

Genome refers to the basic set of chromosomes. In a genome, each type of chromosome is represented only once. Now genomics is being developed as a sub discipline of genetics which is devoted to the mapping, sequencing and functional analysis of genomes.

Types of Genomics:
The discipline of genomics consists of two parts, viz. structural genomics and functional genomics.
Structural Genomics : It deals with the study of the structure of entire genome of a living organism. In other words, it deals with the study of the genetic structure of each chromosome of the genome. It determines the size of the genome of a species in mega-bases [Mb] and also the genes present in the entire genome of a species.

Functional Genomics: The study of function of all genes present in the entire in the genome is known as functional genomics. It deals with transcriptome and proteome. The transcriptome refers to complete set of RNA’s transcribed from a genome and proteome refers to complete set of proteins encoded by a genome.

Question 4.
(a) What are the basic facilities that should be available for tissue culture in a biotechnology laboratory ? [4]
(b) Explain the experiment which proves the semi-conservative mode of replication. [4]
(c) What is cDNA ? [2]
Answer:
(a) A biotechnology laboratory is a place for working with a variety of chemicals and desired organisms since several culture media are prepared and organic materials are present, their exists chance for the presence of high spectrum of microbial community.

Facilities and Tools for Tissue culture in a Biotechnology’ Laboratory:
A biotechnology laboratory should be well-equipped with the instruments. Plant tissue culture can be defined as a collection of techniques/methodologies that are employed to grow or maintain plant cells, tissues or organs on a nutrient culture medium under aseptic growth conditions. Micro propagation is a technique which is widely used for the mass production of clones of a plant. Due to its diverse applications in reviving/ maintaining plants this technique has immensely benefited our society. Therefore, it becomes important to understand the requirements to start tissue culture laboratory.

Some of the prerequisite basic facilities for any laboratory, which actively practices the plant tissue culture methods to grow plants, are :

• Washing area
• Glassware
• Autoclave
• Areas for media preparation, sterilization, and storage
• Area for transfer of cultures/medium in aseptic conditions.
• Environmentally controlled incubators or culture rooms.

Biotechnology equipment available for all major research laboratory’ techniques, including:

• Electrophoresis and blotting.
• Imaging and quantitation.
• Real-time PCR and digital PCR.
• Transfection.
• Flow cytometry, cell sorting, and cell counting.
• Preparative and analytical chromatography.
• Spectrophotometry and fluorometry.

(b) In 1958, for the first time M.S. Messelson and F. W. Stahl put experimental evidences for replication of DNA in a semi-conservative way as proposed by Waston and Crick (Fig). They grew the cells of Escherichia coli in the medium containing heavy’ isotopic nitrogen (¹⁵N) for about 14 cell generations. This was done with the objective that all light isotope nitrogen (¹⁴N) of purine and pyrimidine could be replaced by the isotopic heavier nitrogen i.e., ¹⁵N. The cells were taken out the DNA of cells was removed. The cells grown on ¹⁵N containing medium were filtered and properly w’ashed. Density of ¹⁵N – DNA w as heavier (1.772 g/cc) as compared to normal DNA (1.708 g/cc).

Again the cells grown on ¹⁵N containing medium were taken out, washed properly and grown on medium containing light isotopic nitrogen (¹⁴N) for one to two generations. The generation time of E.coli is 30 minutes. Therefore, it was possible to remove cells after 30 minutes or 60 minutes. Density of ¹⁴N-DNA of such cells was measured by following density gradient centrifugation. Explanation of the result obtained by Messelson and Stahl can be summarized as below:

After 14 generation all the ¹⁴N of bases were replaced by ¹⁵N. This is why the density of ¹⁵N DNAwas higher.

DNA sample analysed after one cell generation transferred to ¹⁴N – containing medium showed only one density band when observed under ultra violet light. The DNA contained 50% each of ¹⁵N and ¹⁴N and, therefore, the density 7 was intermediate between the density of heavy DNA and light DNA.

DNA analysed after two generations of E. coli cells showed the presence of two bands due to types of DNA helix. One type consisted of ¹⁵N – ¹⁵N hybrid and the second were made- up of only ¹⁵N/.e., ¹⁴N- ¹⁴N. The ratio of heavy and light DNAwas 3 : 1. Moreover, the ratio of heavy DNA is subsequent generations gradually decreased and that of light DNA increased. This experiment shows that DNA replicates by semi-conservative method. Thereafter, several evidences for semi-conservative way of DNA replication were given by many scientists such as J. Cairns (1962) and J.HTylor (1957) using auto radigraphic techniques is E. coli and Vicia fab a. respectively

(c) cDNA : The double stranded molecules prepared by using mRNA as template and reverse transcriptase are called cDNA. These are expressed genes of the genomic DNA. By using cDNA molecules, substantial number of sequences have been determined and deposited in database.

Question 5.
(a) Explain any four methods employed to induce haploid production. [4]
(b) Describe the automated method of DNA sequencing. [4]
(c) What is the difference between gel electrophoresis and gel permeation? [2]
Answer:
(a) In vitro culture of haploid cells of plants {e.g., pollen grains from anther and ovules from ovary) is possible. In vivo technique of haploid production includes the following :

• Androgenesis: Production of haploid plants by development of an egg cell containing male nucleus. The female nucleus is eliminated before fertilisation.
• Gymnogenesis: Production of haploid plant by the development of an unfertilised egg cells because of delayed pollination (through use of abortive pollen pre-exposed to ionising irradiation or using an alien pollen). It is found in interspecific crosses of potato.
• Chemical Treatment: Some chemicals like chloramphenicol and parafluorophenylalanin may induce chromosome elimination in somatic cells or tissues giving rise to haploid.

In 1953, Tulecke successfully produced callus from isolated pollen culture of gymnosperms. This made a hope to raise haploid plants through pollen culture. For the first time Hameya and Hinata (1970) produced tissues from the isolated pollen grains of an angiosperm.

For the first time S.Guha and P.Maheshwari (1964) produced haploid embryos in vitro from isolated anthers of Datura innoxia. Haploid production has immense use in plant breeding and improvement of crop plants. Haploids provide an easier system for induction of mutation. They can be employed for rapid selection of mutants having traits for disease resistance. The Institute of Crop Breeding and Cultivation (China) has developed the high yielding and blast resistant varieties of rice zhonghua No. 8 and zhonghua No. 9 through transfer of desired alien gene.

(b) Automatic DNA Sequencers: Automatic sequencing machines were developed during 1990’s. It is an improvement of Sanger’s method. In this new method, a different fluorescent dye is tagged to the aWNTPs. Using this technique, a DNA sequence containing thousands of nucleotide’s can be determined in a few horns. Each dideoxynucleotide is linked with a fluorescent dye that imparts different colours to all the fragments terminating in that nucleotide. All four labelled ddNTP’s are added to a single capillary tube. It is a refinement of gel electrophoresis which separates fastly. DNA fragments of different colours are separated by their size in a single electrophoretic gel.

A current is applied to the gel. The negatively charged DNA strands migrate through the pores of gel towards the positive end. The small sized DNA fragments migrate faster and vice-versa. All fragments of a given length migrate in a single peak. The DNA fragments are illuminated with a laser beam. Then the fluorescent dyes are excited and emit light of specific wavelengths which is recorded by a special ‘recorder’. The DNA sequences are read by determining the sequence of the colours emitted from specific peaks as they pass the detector. This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by the computer. Colour of each dya represents the different nucleotides. Computer converts the data of emitted light in the nucleotide sequences.

(c) Gel electrophoresis is a technique by which negatively charged DNA fragments are separated by forcing them to move towards the anode under an electric field through a medium/matrix. Gel permeation or filtration involves that molecules of different sizes can be separated from each other on the basis of their ability to enter the pores within the beaded gel, followed by passing down a column containing the gel. The technique is used in protein purification.

Question 6.
(a) What is in vitro pollination ? Why is it done ? Write the steps involved in this process. [4]
(b) Why is Agrobacterium called a natural genetic engineer ? How does it help in creating transgenic plants ? [4]
(c) Write a short note on site directed mutagenesis. [2]
Answer:
(a) In vitro Pollination : Cultivation of plant tissue or other organs on artificial media in a test tube or conical flask is called in vitro technique. The process of seed formation following stigmatic pollination of cultured pistil has been referred to as in vitro pollination.

Need of Invitro Pollination:

• For the production of homozygous plant
• For the conservation of extinct plant species
• Hybrid production
• Reducing the breeding cycle
• Overcome the dormant period
• Production of haploid plant
• Conservation of germplasm

(b) The bacterium Agrobacterium tumefaciens contains inside it s cell, a plasmid, i.e., an extrachromosomal self-replicating small DNA, known as Ti plasmid (tumour including plasmid) that induce overproduction of growth factors in the host cells iventually leading to cell proliferation and tumour formation. This bacterium establishes contact with suitable root cells of plants by recognising chemical signals and then prepares it’s plasmid ready to be delivered through t-pilus developed by type IV secretion system. Then it transfers this DNA (also known as t-DNA) into the plant host cell. This delivery requires assistance of several virproteins.

The t-DNA is transferred and stably integrated into the host chromosome which later wards leads in tumour development in the host. The whole process of successful DNA transfer and integration is performed and monitored by the bacteria itself (which in vivo is done by genetic engineers in laboratories). That’s -why Agrobacterium tumefaciens is called the Natural genetic engineers of plants.

Role of Agrobacterium tumefaciens in the production of Transgenic Plants:
Genetically Modified Organisms (GMO’s) are organisms which are formed when the gene of interest is specifically inserted into the host organisms in order to alter its genome and produce an organism with the required desirable traits.

Agrobacterium tumefaciens is a bacterium which naturally causes tumors in plants which is caused by the transfer of DNA (Deoxyribonucleic Acid) from the bacterium to the plant it infects.

Therefore, in the production of transgenic plants by A. tumefaciens, plants do not acquire different traits as A. tumefaciens is used to transfer genes so that the plants acquire the traits. Also, neither resistance is given to plants by this bacterium nor plant genes are incorporated into the genome of this bacterium in the production of transgenic plants.

(c) Site – Directed Mutagenesis: In nature, mutations arise spontaneously but they are rare such as sickle cell anemia. The most powerful method of introducing point mutation into a gene is known as Oligonucleotide or site-directed mutagenesis.

Change in a single nucleotide base pair is called “point mutation”. Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a specific site in the DNA molecule. Using this technique specific point of a gene can be mutated. Therefore, this method has been used to understand the function of many genes. Moreover, this technique can only be used when nucleotide sequence of gene is known.

Question 7.
(a) What is HGP ? Name any two scientists involved in this. Write any two achievements of HGP. [4] (h) List the functions of the following in Bioinformatics [4]
(i) ENTREZ
(ii) PDB
(iii) FASTA
(iv) MGD
(c) Mention two differences between the organisation of prokaryotic and eukaryotic genomes. [2]
Answer:
(a) (i) HGP: HGP means Human Genome Project. It was officially started on October. 1990 in the USA. This is an International Scientific Research Project with a primary goal to determine the sequence of chemical base pairs which make-up DNA and to identify approximately 25000 genes of the human genome from both a physical and functional standpoint.

Significance of genomics was substantially increased when the Human Genome Project was conceived. Research work on Human Genome Project was successfully completed only due to international collaboration involving 60 countries, 20 Genome Research Centers and more than 1000 scientists.

The US side of the Human Genome Project was initially led by James Watson (one half of Crick and Watson, who discovered the structure of DNA), and later by Francis Collins. John Sulston, who was the director here at the Welcome Trust Sanger Institute (at that time called the Sanger Center), was principal leader of the UK side of the project.

Achievements of HGP:
The main objective of this project was to find the full structure of human genes. After that the human genome project kept the target in front of it to know about the protein made by human genes. As a result it would be possible to find out the presence, decreasing number and faults of genes and likewise it would be possible to develop the process of treatment of the diseases like cancer, diabetes, AIDS etc. Along with this, the progress in the treatment technique is also and objective of this project.

The greatest achievement of this project is that scientists can find out the structure of human genes and successfully trace the structure of genes. It has been possible to develop the technique regarding the treatment of defective genes only because of the human genome project.

The treatment of the hereditary’ diseases has been easier because of this project. DNA interference is recently developed technique regarding treatment by which it would be possible to treat many uncureable diseases. Along with this, the development of human, phy sical and mental structure etc., are the main achievements of human genome project.

(b) (i) ENTREZ: Integrated information database retrieval system of NCBI. Using Entrez system we can access literature, sequences (both proteins and nucleotides) and structures (3D).

(ii) PDB: (Protein Data Bank): This database has sequence of those protein whose 3-D structures are known Sources: NCBI – USA; EBI, U.K.

(iii) FASTA:

• FASTA is a DNA and protein sequence alignment software package.
• The current FASTA package contains programs for protein: protein, DNA: DNA, protein: translated DNA (with frame shifts), and ordered or unordered peptide searches. Recent versions of the FASTA package include special translated search algorithms that correctly handle frame shift errors (which six-frame-translated searches do not handle very well) when comparing nucleotide to protein sequence data.

(iv) Mouse Genome Database:
MGD strives to provide an extensively integrated information resource with experimental details annotated from both literature and on-line genomic data sources.

MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse.

(c)

Question 8.
(a) Briefly describe the steps involved in the Southern blotting technique. [4]
(b) What is the need of germplasm conservation ? Give an account of the in-situ and ex-situ conservation of germplasm. [4]
(c) What is peptidoglycan ? Where is it found ? [2]
Answer:
(a) Southern blotting is a technique for transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon membrane, and is carried out prior to detection of specific molecules by hybridization probing.

In this technique, DNA is usually converted into conveniently sized fragments by restriction digestion and separated by gel electrophoresis, usually on an agarose gel. The DNA is denatured into single strands by incubation by alkali treatment.

The DNA is transferred to a nitrocellulose filter membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation they had on the gel. This process is called blotting.

The nitrocellulose membrane is how removed from the blotting stack.

The blot is incubated with many copies of a radioactive probe which is single-stranded DNA. This probe detect and identify base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g., alkaline phosphatase or horseradish peroxidase). This step is known as hybridisation reaction.

The location of the probe is revealed by incubating it with a colourless substrate that the attached enzyme converts to a coloured product that can be seen or gives off light which will expose X-ray film. If the probe was labelled with radioactivity, it can expose X-ray filmdirectly. The images of radioactive probe are revealed as distinct bands on the developed X-ray film.

(b) Germplasm Conservation: The sum total of all the genes present in a crop plant and its related species is called germplasm . The wild and traditional species must be preserved through germplasm conservation.

Importance or Need of Germplasm Conservation:
Due to rapidly growing human population, plant and agriculture scientists have produced many new varieties to feed the population. Because the traditional and wild varieties cannot meet the demand of increasing population. But the traditional varieties need to be preserved because they are important for future breeding programmes. However, there is danger of erosion of genetic resources due to extensive use of newly introduced varieties.

In 1972, conservation of habitats rich in genetic diversity was recommended in the UN conference. Then an International Board for Plant Genetic Resource (IBPGR) was established. This board has objectives to provide necessary support for collection, conservation and utilisation of plant genetic resources from anywhere in the world. Germplasm preservation using cell and tissue cultures is done with the following objectives :

• Conservation of somaclonal and gametoclonal variation in cultures,
• Maintenance of recalcitrant seeds,
• Conservation of cell lines producing medicines,
• Storage of pollen for enchancing longevity,
• Conservation of rate germplasm arising through somatic hybridisation.
• Delaying the process of ageing,
• Storage of meristem culture for micropropagation, micrografting and production of disease- free plants,
• Conservation of plant materials from endangered species,
• Establishment of germplasm bank, and
• Exchange of information and germplasm at international level.

Modes of Conservation : It has been estimated that about 9,000 wild plant species are threatened. Global climatic changes also affect the natural plant habitats, thereby contributing to rapid changes in agricultural strategies. There are two modes of conservation of plant species as given below:

(a) In situ Conservation : Since 1980, in situ conservation has received high priority in the world conservation strategy. The method of conservation is to preserve land races with wild relatives in which genetic diversity exists.

(b) Ex situ Conservation: It is the chief mode of conservation of genetic resources including both cultivated and wild ones. Under suitable conditions genetic resources are conserved for a long term as gene bank.

(c) Peptidoglycan : Peptidoglycan, also called murein, is a polymer that makes up the cell wall of most bacteria. It is made-up of sugars and amino acids, and when many molecules of peptidoglycan joined together, they form an orderly cry stal lattice structure. Peptidoglycan is the main component of the Cell wall in most bacteria. Cross-linking between amino acids in the layer of peptidoglycan forms a strong meshlike structure that provides structure to the cell. Peptidoglycan provides a very important role in bacteria because bacteria are unicellular; it gives strength to the outer structure of the organism.

Question 9.
(a) How are biomolecules separated by the following techniques : [4]
(i) Ion exchange chromatography
(ii) Partition chromatography.
(b) What is the cause and the symptoms of the following diseases : [4]
(i) Sickle cell anaemia
(ii) Alkaptonuiria
(c) What is the difference between peptide bond and phosphodiester bond ? [2]
Answer:
(a) (i) Ion Exchange Chromatography: It is based on reversible exchange of ions in solution with ions electrostatically bound to some sort of insoluble support medium. Separation is obtained since different molecules have different degree of interaction with ion exchanger due to difference in their charges, charge densities and distribution of charge on their surface.

(ii) Partition Chromatography: Partition chromatography is the process of separation whereby the components of the mixture get distributed into two liquid phases due to differences in partition coefficients during the flow of mobile phase in the chromatography column. Here, the molecules get preferential separation in between two phases, i.e., both stationary phase and mobile phase are liquid in nature. So molecules get dispersed into either phases preferentially. Polar molecules get partitioned into polar phase and vice-versa. This mode of partition chromatography applies to liquid-liquid, liquid-gas chromatography and not to solid-gas chromatography. Because partition is the phenomenon in between a liquid and liquid or liquid and gas or gas and gas. But not in solid involvement.

(b) (i) Sickle cell anaemia : It is an autosomal hereditary disorder due to a mutation of single nitrogen base. It results in the formation of an abnormal haemoglobin called haemoglobin S (Hb^s). In this, only one amino acid-6th amino acid of β-chain glutamic acid is replaced by valine. The erythrocytes become sickle shaped under oxygen deficiency as during strenous exercise and at high altitudes. They cannot pass through narrow capillaries. They clog blood capillaries. Blood circulation and oxygen supply is disturbed. Spleen and brain get damaged. Patient feels acute weakness. Homozygotes (Hb^s / Hb^s) usually die before reaching maturity.

(ii) Alkaptonuria: This was one of the first metabolic diseases described by Garrod in 1908. It is an inherited metabolic disorder produced due to deficiency of an oxidase enzyme required for breakdown of homogentisic acid (acid called alcapton, hence, alkaptonuria is also written as alcaptonuria.) Lack of the enzyme is due to the absence of the normal form of gene that controls the synthesis of the enzyme. Hence, homogentisic acid then accumulates in the tissues and is also excreted in the urine. The most commonly affected tissues are cartilages, capsules of joints, ligaments and tendons. The urine of these patients if allowed to stand for some hours in air, turns black due to oxidation of homogentisic acid.

(iii) Peptide bond:
A peptide bond is a covalent bond formed between two amino acids. Living organisms use peptide bonds to form long chains of amino acids, known as proteins. Proteins are used in many roles including structural support, catalyzing important reactions, and recognizing molecules in the environment. A peptide bond is therefore the basis of most biological reactions.

Phosphodiester bond:
A phosphodiester bond occurs when exactly two of the hydroxyl groups in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. Phosphodiester bonds are central to all life on Earth, as they make up the backbone of the strands of nucleic acid.

ISC Class 12 Biotechnology Previous Year Question Papers