Class XII – Biotechnology: Principles and Processes – PYQs

Ans. Salmonella tymphimurium was used in the first instance of the construction of artificial recombinant DNA molecule because it has a plasmid which has an autonomously replicating circular extrachromosomal DNA. It also has an antibiotic resistance gene.

Ans. Stanley Cohen and Herbert Boyer accomplished this in 1972. Antibiotic resistant gene was isolated using restriction enzymes and introduced into the plasmid of the bacterium, i.e. Salmonella tymphimurium.

Later the recombinant plasmid was introduced into the bacterium, E. coli, so that it could make copies of gene.

Ans. No, eukaryotic cells do not have restriction endonucleases because the DNA of eukaryotes is highly methylated due to the presence of enzymes methylase which protects the DNA from the activity of restriction enzymes.

But prokaryotes/ bacteria have this enzyme as a defence mechanism to destroy the foreign DNA or to restrict the growth of bacteriophages. Hence, all the restriction endonucleases have been isolated from various strains of bacteria.

Ans. The convention for naming of restriction endonuclease proceeds in a way that the first letter of the name comes from the genus and the second two letters come from the species of prokaryotic cell from which they were isolated.

For example, in restriction endonuclease, Eco RI comes from E. coli RY13, where the letter ‘R’ is derived from the name of strain. Roman numbers following the names, indicate the order in which the enzymes were isolated from that strain of bacteria.

Ans. Exonucleases cleave base pairs of DNA at their terminal ends (either 5 or 3 ¢ ¢), while the endonucleases cleave DNA at any point within the DNA segment at specific position except terminal ends.

Ans. Restriction endonuclease EcoRI cuts the DNA strands a little away from the palindromic sequences, but between the same two bases on the two strands.

Ans. Five examples of palindromic DNA sequences are

(i) 5’–A A G C T T–3’

3’–T T C G A A–5’

(ii) 5’–G G A T C C–3’

3’–C C T A G G–5’

(iii) 5’–A G G C C T–3’

3’–T C C G G A–5’

(iv) 5’–G A A T T C–3’

3’–C T T A A G–5’

(v) 5’–A C T A G T–3’

3’–T G A T C A–5’

Ans. Restriction enzymes cut the strands of the DNA, a little away from the centre of the palindromic sites, but between the same two bases on opposite strands. This leaves sticky single-stranded position at the ends. These overhanging stretches are called ‘sticky ends’. These are named so because they form hydrogen bonds with their complementary cut counter parts which facilitates the action of the enzyme DNA ligase.

Ans. All cloning vectors have a selectable marker. Selectable marker helps in identifying or selecting transformants and eliminating non-transformants by selectively permitting the growth of the transformants.

Ans. DNA molecules are bigger in molecular size as compared to the enzymes. Small segments of the DNA called genes, control the synthesis of proteins and enzymes. DNA is made up of sugar, phosphate and nitrogen, while enzymes are made up of only protein molecules.

Ans. The restriction enzymes are known as molecular scissors.

These are used to cut plasmid DNA as well as foreign DNA at desired sites. The foreign DNA is then inserted into plasmid DNA. Then the plasmid takes the foreign DNA into the desired host organism.

Example Restriction enzyme Hind II. It was isolated from Haemophilus influenzae.

It produces DNA segments with blunt ends.

Ans. These are extrachromosomal, self-replicating, double-stranded, closed and circular DNA molecules, found only in bacteria and few yeast cells. These are used as vectors to carry the desired gene (foreign genes) into the desired organisms.

Example pBR322 is widely used plasmid vector. This plasmid has genes for resistance against ampicillin and tatracycline.

Ans. The recombinant DNA can multiply as many times as the copy number of the plasmid vector, thereby determining the yield of recombinant protein. So, higher the copy number of the plasmid vector, higher will be the yield of recombinant protein.

11. Explain briefly.

i) Restriction enzymes and DNA

Ans. These enzymes cut the helix of DNA on a specific site. So, these are also known as molecular scissors. DNA is a genetic material which is responsible to transfer genetic characters from one generation to the other.

Ans. It is an enzyme used to break the cell wall of fungi to release its cellular parts. Certain plants and fungus consist of a cell wall composed of chitin, which provides strength to the cell. Chitinase is used when there is a need to break this wall and allows entry of foreign DNA in plant cells.

Ans. Coding sequence of β-galactosidase is a better selectable marker, as the recombinants and non-recombinants are differentiated on the basis of their ability to produce colour in the presence of a chromogenic substrate, while the selection of recombinants due to inactivation of antibiotic resistant gene is a tedious and time taking process to grow them simultaneously on two antibiotics.

Ans. The recombinant/transformants can be selected out from the non-recombinants/ non-transformants by plating the transformants on ampicillin-containing medium. The transformants will grow in it, while the non-transformants will not grow.

Ans. It acts as a selectable marker

14. What modification is done on the Ti plasmid of Agrobacterium tumefaciens to convert it into a cloning vector?

OR

State how has Agrobacterium tumefaciens been made a useful cloning vector to transfer DNA to plant cells.

Ans. Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is modified into a cloning vector by removing/altering the gene responsible for its pathogenic property. This is so, that it does not cause harm, but can act as a delivery system for the genes of interest into a variety of plants.

Ans. CaCl₂ is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. The divalent Ca²⁺ ions create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells.

Ans. DNA fragments formed by the use of restriction endonucleases are separated by gel electrophoresis.

Ans. Since, the DNA fragments are negatively charged molecules, so they can be separated by applying an electric field which makes DNA molecules move towards the anode (+), under an electric field through an appropriate medium or matrix. The most commonly used matrix is agarose. DNA fragments resolve (separate) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, farther it moves. The separated DNA fragments can be visualised only after staining the DNA with Ethidium Bromide (EtBr) followed by exposure to UV radiation.

These separate stained bands of DNA are cut out from the agarose gel and extracted from the gel piece, by a process known as elution.

Ans. Plasmid DNA when cut and separated on agarose gel electrophoresis, shows one DNA band because plasmid is a circular DNA molecule and when it is cut with enzyme, it becomes linear, but does not get fragmented. Whereas, a linear DNA molecule gets cut into two fragments. Hence, a single DNA band is observed for plasmid, while two DNA bands are observed for linear DNA in agarose gel.

Ans. Amplification of gene is done using Polymerase Chain Reaction (PCR). It is carried out in the following steps

(i) Denaturation: The double-stranded DNA is denatured by applying high temperature of 95°C for 15 seconds. Each separated strand acts as a template.

(ii) Annealing: Two sets of primers are added, which anneal to the 3¢ end of each separated strand. This step is carried out at a slightly lower temperature (40°-60°C).

(iii) Extension: DNA polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction. Taq polymerase is used in the reaction, which can tolerate heat. All these steps are repeated many times to get several copies of the desired DNA.

Ans. Bacterium, Thermus aquaticus is a source of enzyme Taq polymerase. As it is a thermostable enzyme and works at high temperature, it is used to amplify DNA in vitro by PCR. The amplified fragment of desired DNA can be used to ligate with the vector for further cloning.

Ans. If denaturation of double-stranded DNA does not take place, then primers will not be able to anneal to the template. Amplification will not occur and no extension will take place.

Ans. (i) Role of heat In PCR (in vitro), the DNA strands are separated by heating at 95°C for 15 seconds. Heating causes the H-bonds between bases of two strands to get broken leading to unwinding.

(ii) Role of primers Primers are short lengths of DNA about 20 bp long that are required to start DNA polymerisation in PCR. The primers hybridise to their complementary sequence on the DNA strands at 40-60°C temperature and help in DNA polymerisation.

(iii) Role of Thermus aquaticus An enzyme called Taq polymerase is isolated from Thermus aquaticus. Since, this bacterium thrives in temperature as high as 95°C, without undergoing denaturation. Therefore, this enzyme is used in PCR instead of normal DNA polymerase.

Ans. Three uses of PCR are

(i) It is used during rDNA for the production of newer and desired DNA.

(ii) It is used for DNA sequencing.

(iii) It is used in DNA fingerprinting.

Ans. Besides better aeration and mixing properties stirred-tank bioreactors also facilitate mixing and oxygen availability throughout the bioreactor. It has an oxygen delivery system, a foam control system and a temperature controller. Small volumes of cultures are periodically withdrawn from the reactor for sampling.

Ans. The cells can be multiplied in a continuous culture system. In this, the used medium is drained out from one side, while fresh medium is added from the other side to maintain the cells in their physiologically most active (log/exponential) phase. This type of culturing method produces a larger biomass leading to higher yields of desired products.

Ans. (i) Differences between plasmid and chromosomal DNA are as follows

Ans. The desired gene is cut by using the enzymes restriction endonucleases. Firstly, the restriction endonucleases that recognise the palindromic nucleotide sequence of the desired gene is identified.

The endonuclease inspects the entire DNA sequences to find and recognise the site. It cut each of the double helix at a specific point, which is a little away from the centre of the palindromic site. The cutting site is between the same two bases on the opposite strands. This results in overhanging of single-stranded stretches, which act as sticky ends.

It can be represented by the following diagram

Ans. Multiple copies of the desired gene are synthesised by Polymerase Chain Reaction (PCR) method. In this method, the desired gene is synthesised in vitro. The double-stranded DNA is denatured using high temperature of 95° C and the strands are separated. Each separated strand acts as a template. Two sets of oligonucleotide primers are annealed to the denatured DNA strands. The thermostable Taq polymerase extends the primers using nucleotides provided in the reaction mixture. Finally, the amplified fragments are ligated into recipient cells.

Ans. Bioreactors are the large volume vessels approximately (100-1000 L) in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial, plant, animal or human cells.

A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions like temperature, pH, substrate, salts, vitamins and oxygen. The cells can also be multiplied in a continuous culture system. In this, the used medium is drained out from one side while fresh medium is added from the other side to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein. Thus, it plays a very important role particularly in traditional biotechnology.

Ans. The most commonly used bioreactor is simple stirred tank bioreactor. It is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer activity facilitates even mixing and availability of O₂ throughout the bioreactor.

Alternatively, air can also be bubbled into the medium. As shown in the figure below, it has an agitator system, a temperature control system, a pH control system and sample ports so that small samples can be withdrawn periodically.