ISC Biotechnology 2015 Class-12 Previous Year Question Papers Solved

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.
ISC Biotechnology Question Paper 2015 Solved for Class 12 5
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.  (ISC Biotechnology 2015 Class-12)
(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 5:
(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.  (ISC Biotechnology 2015 Class-12)
(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 6:
(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.
ISC Biotechnology Question Paper 2015 Solved for Class 12 6
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.  (ISC Biotechnology 2015 Class-12)
(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 7:
(a)

Name Class Location
(i) Cellulose Polysachharide Cell wall
(ii) Histone Basic proteins Chromosome (Eukaryotes)
(iii) rRNA Nucleic Acid Ribosome
(iv) Cholestrol Steroids (Derived lipids) Produced in liver in human beings animals and some plants (e.g., potato)

(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.  (ISC Biotechnology 2015 Class-12)
(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 8:
(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.

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