Very Short Answer Type Questions

Question. Write the names of the enzymes that are used for isolation of DNA from bacterial and fungal cells respectively for Recombinant DNA Technology
Answer : Lysozyme for bacterial cells, chitinase for fungal cells. 

Question. How can bacterial DNA be released from the bacterial cell for biotechnology experiments ?
Answer : (Breaking the cell open) Treating with lysozyme.

Short Answer Type Questions

Question. How is insertional inactivation of an enzyme used as a selectable marker to differentiate recombinants from non-recombinants ?
Answer : The presence of chromogenic substrate gives blue coloured colonies, in presence of β-galactosidase.
Presence of an insert (recombinant DNA) results into inactivation of the enzyme, colonies with inactivation of β-galactosidase do not produce any colour.
Detailed Answer :
The insertion of rDNA into the coding sequence of an enzyme alpha-galactosidase leads to the inactivation of the enzyme called insertional inactivation. The recombinants do not produce a blue coloured colonies in the presence of chromogenic substrate while the non-recombinants produce a blue colour.

Question. Name the type of bioreactor shown. Write the purpose for which it is used.

Answer : Simple stirred tank bioreactor. Large scale production of recombinant protein / Raw materials are biologically converted into specific products or enzymes, using microbial plants / animals / human cells.

Question. What is a primer ? What is its role in PCR ?
Answer : A primer is a small segment of DNA that binds to a complementary strand of DNA.
Primers are necessary to start the functioning of DNA polymerase enzyme and therefore, are necessary in polymerase chain reaction. 

Question. Why is the ‘insertional inactivation’ method to detect recombinant DNA preferred to ‘antibiotic resistance’ procedure? 
Answer : The presence of a chromogenic substrate gives blue coloured colonies in absence of an insert / in non-transformants, presence of an insert (in the enzyme site), results into (insertional inactivation of theβ-galactosidase) colonies which do not produce colour. 
Antibiotic resistance method requires duplicate plating / cumbersome procedure.

Question. Why is Taq polymerase preferred to PCR? Mention the source of this enzyme ?
Answer : Taq polymerase is used for amplification of DNA / gene, (usually enzymes get denatured). Taq polymerase is thermostable, remains active at high temperature. It is obtained from Thermus aquaticus. 
Detailed Answer :
Thermostable Taq polymerase extends the primers using nucleotides provided in the reaction and genomic DNA as template.
The source of this enzyme is Thermus aquaticus. 

Question. Write the functions of the following in biotechnology.
(i) Polymerase chain reaction technique
(ii) Restriction endonucleases
(iii) Bacterium Thermus aquaticus.
Answer : (i) Multiple copies of gene of interest can be obtained.
(ii) They can cut DNA molecule at a particular point by recognizing a specific sequence of base pairs. Thus they are useful in forming recombinant DNA.
(iii) Thermus aquaticus is the source of Taqpolymerase which remains active during high temperature induces denaturation of DNA in PCR technique and therefore allows chain reaction to proceed.

Question. (a) How has the development of bioreactor helped in biotechnology?
(b) Name the most commonly used bioreactor and describe its working?
Answer : (a) Larger biomass / large volume of culture can be processed leading to higher yields of desired specific products (protein / enzymes), under controlled condition
(b) Stirring type 
♦ Mixing of reactor contents evenly (with agitator system or a stirrer). 
♦ Facilitates oxygen availability. 
♦ Temperature / pH / foam control under optimum conditions. 

Question. List the key tools and steps used in recombinant DNA technology. 
Answer : Tools are enzymes, vehicle DNA, passenger DNA.
Recombinant DNA technology involves the following steps:
(i) Isolation of DNA.
(ii) Fragmentation of DNA by restriction endonucleases.
(iii) Isolation of the desired DNA fragment.
(iv) Amplification of the gene of interest.
(v) Ligation of the DNA fragment into a vector using DNA ligase.
(vi) Transfer of recombinant DNA into the host.
(vii) Culturing the host cells on a suitable medium on a large scale.
(viii) Extraction of the desired product.
(ix) Downstream processing of the product as a finished product ready for marketing. 

Question. (i) Why is Taq polymerase used instead of ordinary DNA polymerase in polymerase chain reaction (PCR) ? Name the source organism of Taq polymerase.
(ii) What is PCR used for ?
Answer : (i) It is thermostable / remains active during the high temperature induces denaturation of (double stranded) DNA, (bacterium) Thermus aquaticus.
(ii) To obtain multiple copies of the gene (or DNA) of interest. 
Detailed Answer :
(i) Taq polymerase is used in PCR instead of ordinary DNA because of its thermostable nature. It remains active at high temperature during the denaturation of double stranded DNA. The source organism of Taq polymerase is Thermus aquaticus.
(ii) Polymerase chain Reaction (PCR) is used to obtain multiple copies of the gene of interest.

Question. Write the steps you would suggest to be undertaken to obtain a foreign-gene-product.
Answer : Insert a piece of alien or desired or foreign DNA into a cloning vector, transfer it into a Bacterial / plant / animal cell, the alien DNA gets mutiplied, optimised condition (temperature pH, substrate, salts, vitamins, O₂) provided to the culture / culture in bioreactor / in continuous culture system to induce the expression of the target product, extracting the desired product, purifying it by using different separation techniques. 
Detailed Answer :
(i) Insertion of a piece of desired DNA into cloning vector to get recombinant DNA.
(ii) Transfer of recombinant DNA into a host cell.
(Plant or animal or bacterial cell).
(iii) The alien DNA will get multiplied.
(iv) After the cloning of gene of interest, optimised conditions are provided to the culture to induce the expression of the target gene.
(v) Extraction of the desired product.
(vi) Purification of desired products by using different separation technique.

Question. ‘‘A very small sample of tissue or even a drop of blood can help determine paternity’’. Provide a scientific explanation to substantiate the statement.
Answer : The above statement can be substantiated through DNA finger printing by southern blot method and by PCR.
DNA from all the cells of an individual shows the same degree of polymorphism.
The polymorphs are heritable. An individual inherits 50% of the chromosomes from maternal and 50% from the paternal parent.
Small amount of DNA from blood or tissue is taken and amplified by PCR, which can be used in DNA finger printing so as to identify the paternity. 

Long Answer Type Questions

Question. Diagrammatically explain the steps of rDNA technology. 
Answer : 

Question. If a desired gene is identified in an organism for some experiments, explain the process of the following :
(i) Cutting this desired gene at specific location
(ii) Synthesis of multiple copies of this desired gene. 
Answer : (i) (a) Identifying the restriction endonuclease that recognises the palindromic nucleotide sequence of the desired gene.
(b) The restriction endonuclease inspects the DNA sequences – finds and recognises the site.
(c) Cuts each of the double helix at the specific point – a little away from the centre of the palindromic site – between the same two bases on the opposite strand.
(d) Makes the over hanging stretch single stranded portion as a sticky end.
(ii) (a) By PCR / Polymerase Chain Reaction.
(b) Desired gene is synthesised in vitro.
(c) DNA is denatured – Annealed using two sets of primers.
(d) Thermostable Taq polymerase extends the primers using nucleotides (provided in the reaction and genomic DNA as template).
(e) Amplified fragments are ligated.