Principles of Biotechnology and Tools of Recombinant DNA Technology
Very Short Answer Type Questions
Question. Suggest a technique to a researcher who needs to separate fragments of DNA.
Answer : Gel electrophoresis is used to separate DNA fragments.
Question. How can following be made possible for biotechnology experiments?
(i) Isolation of DNA from bacterial cell.
(ii) Reintroduction of recombinant DNA into a bacterial cell.
Answer : (i) By treating the cell with the enzyme lysozyme.
(ii) By making the bacterial cell competent.
Question. Name the host cells in which micro–injection technique is used to introduce an alien DNA.
Answer : Animal cell.
Question. How is the action of normal endonuclease enzymes different from that of restriction endonuclease ?
Answer : Normal endonuclease cuts at random position within a DNA sequence, whereas restriction endonuclease recognizes and cut specific nucleotide sequences within DNA.
Question. Write the two components of the first artificial recombinant DNA molecule constructed by Cohen and Boyer.
Answer : Restriction enzyme and vector.
Question. Why EtBr is used in gel electrophoresis in spite of it being highly carcinogenic ?
Answer : EtBr is an intercalating agent. It stacks itself in the DNA bases, and fluoresce under U.V light, thus helps in identification of DNA.
Question. State what happens when an alien gene is ligated at the Sal I site of pBR322 plasmid.
Answer : When an alien gene is ligated at the Sal I site of tetracycline resistance gene in the vector pBR322, the recombinant lose tetracycline resistance due to insertion of the foreign DNA.
Question. State what happens when an alien gene is ligated at Pvu I site of pBR322 plasmid.
Answer : When an alien gene is ligated at the Pvu I site of ampicillin resistance gene in the vector pBR322, the recombinant plasmids lose ampicillin resistance due to insertion of the foreign DNA.
Question. Name two enzymes that are essential for constructing a recombinant DNA.
Answer : Restriction enzymes / polymerase enzymes / ligase
Question. Why it is not possible for an alien DNA to become part of a chromosome anywhere along its length and replicate normally ?
Answer : Alien DNA must be linked to ori / origin of replication / site to start replication.
Question. Mention the role of Restriction Enzymes in Recombinant DNA technology.
Answer : To cut DNA at specific sites / Molecular scissors (DNA).
Question. Which main technique and instrument is used to isolate DNA from a plant cell ?
Answer : Centrifugation and centrifuge.
Question. Name the material used as matrix in gel electrophoresis and mention its role.
Answer : Agarose is the most commonly used matrix in DNA gel electrophoresis. It provides sieving effect for separation of DNA fragments according to their size.
Question. Why do DNA fragments move towards the anode during gel electrophoresis ?
Answer : DNA fragments are negatively charged.
Question. Write the palindromic sequence that EcoRI recognises.
Answer : Palindromic sequence is asked, write both the sequence with polarity.
Question. Name the specific sequence of DNA in a plasmid that the gene of interest ligates with to enable it to replicate.
Answer : Origin of replication (Ori).
Question. Mention the use of gel electrophoresis in biotechnology experiments.
Answer : Cut fragments of DNA can be segregated / separated.
Question. Mention the type of host cells suitable for the gene guns to introduce an alien DNA.
Answer : Plant cells.
Question. Write the two components of the first artificial recombinant DNA molecule constructed by Cohen and Boyer.
Answer : The two components are antibiotic resistant gene and plasmid vector of Salmonella typhimurium.
Question. Name the technique that is used to alter the chemistry of genetic material (DNA, RNA) to obtain desired result.
Answer : Genetic Engineering / Biochemical Engineering / Biotechnology.
Question. Why is it essential to have ‘selectable marker’ in cloning vector.
Answer : Selectable marker helps in the identification and elimination of non-transformants and permitting the growth of the transformants. Therefore, they are considered essential in cloning vector.
Question. Why is ’plasmid’ an important tool in biotechnology experiments ?
Answer : Plasmids are commonly used to multiply or express particular genes and act as vectors to transfer piece of foreign DNA attached to them.
Question. Explain giving reasons why an alien piece of DNA needs to be integrated to a specific sequence of host DNA for its cloning ?
Answer : Ori (origin of replication) is the specific DNA sequence where the replication of DNA is initiated.
Therefore for multiplication of alien DNA in the host it has to be integrated to the ori (origin of replication).
Short Answer Type Questions – l
Question. Explain palindromic nucleotide sequence with the help of a suitable example.
Answer : Palindrome in DNA is a sequence of base pairs that reads the same on two strands when orientation of reading is the same.
Example : 5’ …. GAATTC …. 3’
3’……CTTAAG …. 5’ 2
Question. Explain with the help of a suitable example the naming of a restriction endonuclease.
Answer : EcoRI.
The first letter of the name comes from the genus and the next two from the name of the species of the bacterium i.e. prokaryotic cell. Thus Eco stands for the genus and species of the prokaryotic cell from which the enzyme was isolated i.e. E. coli R stands for strain.
‘I’ follows order in which enzyme was isolated.
Question. Why are molecular scissors so called ? Write their use in biotechnology.
Answer : The restriction enzymes are known as molecular scissors as they cut the DNA at specific sites or locations.
They help (in genetic engineering) to form recombinant molecules of DNA, which are composed of DNA from different genomes.
Question. How are ‘sticky ends’ formed on a DNA strand ? Why are they so called ?
Answer : 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.
These overhang stretches are called as sticky ends.
They form hydrogen bonds with their complementary cut counterparts.
Question. How does a restriction nuclease function ? Explain.
Answer : Restriction nuclease cut DNA at specific sites.
Exonuclease cuts DNA at the ends, endonuclease cuts at specific position within DNA.
Restriction endonuclease cuts the DNA at specific palindromic sequence.
Question. Explain the work carried out by Cohen and Boyer that contributed immensely to biotechnology.
Answer : Cohen and Boyer invented the technique of DNA Cloning and conducted first genetic engineering experiments. They conducted experiment on removing plasmid from one bacterial cell and reinserting into another bacterial cell.
Question. Write the role of ‘Ori’ and ‘restriction’ site in a cloning vector pBR322.
Answer : Ori : It is a genetic sequence that acts as the initiation site for replication of DNA. Any fragment of DNA, when linked to the ori region, can be initiated to replicate. Restriction site : It is the recognition site for restriction enzymes (such as EcoRI, Hind III, Pvu I and Bam Hl). Recognition sites are the genetic sequences from where the restriction enzymes cut the DNA.
Question. Explain the role of Ti plasmid in biotechnology.
Answer : (i) The Ti plasmid (tumor-inducing plasmid) of Agrobacterium tumefaciens has been modified (does not cause tumour) and used as a cloning vector. The Ti plasmid integrates a segment of its DNA, termed T-DNA into the chromosomal DNA of its host plant cells.
(ii) The T-DNA plasmid causes tumours. As gene transfer occurs without human effort, the bacterium is known as ‘natural genetic engineer’ of plants. Ti plasmids as vectors, transfer foreign genes of interest into the target cells.
Question. Explain how to find whether E. coli bacterium has transformed or not, when, a recombinant DNA bearing ampicillin-resistant gene is transferred into it.
Answer : The recombinant / transformant may be found out from non-recombinant / non-transformant by plating the transformants on ampicillin containing medium. The transformants growing on ampicillin containing medium are then transferred to tetracycline containing medium. The recombinant will grow on ampicillin containing medium but not on that containing tetracycline. But nonrecombinant will grow on both tetracycline and ampicillin containing media.
Question. State the role of DNA ligase in biotechnology.
Answer : The role of DNA ligase in biotechnology is to join two different restricted fragments of DNA to make recombinant DNA. the DNA fragments are joined from their ends.
Question. List the key tools used in recombinant DNA technology.
Answer : Restriction enzymes / Polymerase enzymes / Ligase enzymes / Vectors / Host organisims / E. coli/ Agrobacterium.
Question. State how has Agrobacterium tumefaciens been made a useful cloning vector to transfer DNA to plant cells.
Answer : Agrobacterium tumefaciens has Ti plasmid. This plasmid is modified into a cloning vector, which is no more pathogenic to host plants and is able to deliver genes of interest.
Question. What is EcoRI ? How does EcoRI differ from an exonuclease ?
Answer : EcoRI is restriction endonuclease enzyme.
Exonuclease removes nucleotides from the ends of DNA.
EcoRI makes cuts at specific position within the DNA.
Short Answer Type Questions – ll
Question. Describe a palindrome with the help of an example.
Answer : A DNA sequence that reads the same, on the two strands from 5’- 3’ direction or 3′ → 5′ direction.
Question. (i) In pBR322, foreign DNA has to be introduced in tetR region. From the restriction enzymes given below, which one should be used and why : Pvul, EcoRI, BamHI
(ii) Give reasons, why the other two enzymes cannot be used.
Answer : (i) Bam HI should be used, as restriction site for this enzyme is present in tetR region.
(ii) Pvu I will not be used as restriction site for this enzyme is present in ampR region (not in tetR ).
EcoRI will not be used, as restriction site for this enzyme is not present in selectable marker tetR .
Question. (i) Name the selectable markers in the cloning vector pBR322. Mention the role they play.
(ii) Why is the coding sequence of an enzyme β-galactosidase a preferred selectable marker in comparison to the ones named above ?
Answer : (i) ampR / ampicillin resistance genes, tetR/ tetracycline resistance gene.
They help in identifying and eliminating nontransformants / non- recombinants and selectively permitting the growth of the transformants / recombinants.
(ii) Simpler process / less cumbersome, in the presence of chromogenic substrate recombinants are colourless and non recombinants are blue in colour.
Question. How does a restriction endonuclease help in DNA recombinant technology ?
Explain the mode of action of EcoRI.
Answer : Restriction endonuclease (EcoRI) inspects length of DNA and recognises specific palindromic nucleotide sequence, binds with DNA, cuts each of the two strands of double helix at specific points.
This leaves the single stranded overhanging stretches at the ends. They are called sticky ends.
They form H-bonds with their complementary cut counterparts. This stickiness facilitates action of DNA ligase, when cut by the same restriction enzyme. The resultant DNA fragments have the same kind of sticky ends and these are joined together by DNA ligase.
Question. Explain with the help of an example the relationship between restriction endonuclease and a palindromic nucleotide sequence.
Answer : Restriction endonuclease recognises a specific palindromic nucleotide sequence, in the DNA molecule. Restriction endonuclease cuts the strand of DNA a little away from the centre of palindromic nucleotide sequence but between the same two bases on the opposite strands, leaving
single stranded portions at the end called sticky ends.
(No mark, if polarity is not shown or if only one strand is shown).
Question. Explain the role of the enzyme EcoRI in recombinant DNA technology.
Answer : EcoRI (Restriction endonuclease) acts as molecular scissors. They serve as tools for cutting DNA molecule at specific palindromic sites, inspects length of DNA and recognises specific palindromic nucleotide sequence, binds with DNA, cuts each of the two strands of double helix at specific points.
Question. (i) Why must a cell be made ‘competent’ in biotechnology experiments ? How does calcium ion help in doing so ?
(ii) State the role of ‘biolistic gun’ in biotechnology experiments.
Answer : (i) (a) To take up the (hydrophilic) DNA from the external medium.
(b) Divalent calcium ions increase the efficiency of the cell to take up foreign DNA through pores in the cell wall.
(ii) To introduce alien DNA into the plant cell by bombarding them with high velocity microparticles (gold or tungsten coated with DNA).
Question. Name and describe the technique that helps in separating the DNA fragments formed by the use of restriction endonuclease.
Answer : Gel electrophoresis.
DNA are negatively charged forced to move towards anode, electric field in agarose gel matrix, separate according to their size / sieving effect, smaller fragments moves faster and farther than the larger.
Question. (i) Differentiate between exons and introns.
(ii) What is a plasmid ? Why is it selected as a vector ?
Answer : (i) Exons are the coding or expressed sequences that appear in mature or processed RNA, introns are intervening sequences that do not appear in mature or processed RNA / Exons are codons that code for amino acid sequence, introns do not code for amino acids.
(ii) Autonomously replicating circular DNA / extra chromosomal DNA, exclusively present in bacteria.
Plasmid is selected as vector because it has ability to replicate in the bacterial cell independent of chromosomal DNA and also has high copy number.
Question. Explain the basis on which the gel electrophoresis technique works. Write any two ways the products obtained through this technique can be utilized.
Answer : Basis of technique : Since DNA fragments are negatively charged, they can be separated by forcing them to move towards the anode under an electric field through a medium of matrix (agarose).
Uses : (i) The purified DNA fragments are obtained by gel electrophoresis.
(ii) They are used in DNA fingerprinting.
Question. How are the following used in biotechnology ?
(ii) recognition sequence.
(iii) gel electrophoresis.
Answer : (i) Plasmids are relatively small DNA sequences that can self replicate and exist independent of the chromosome. Plasmids often carry antibiotic resistance genes that makes them selectable.
They can be genetically modified – cut at specific locations using restriction enzymes and new DNA sequences are included. This recombinant plasmid is reintroduced into bacteria and easily forced to multiply in large numbers at relatively low cost.
Thus, plasmids have been used in biotechnology to develop vectors (tools) for basic research like studying new genes as well as to produce therapeutic chemicals.
(ii) The restriction enzymes cut the DNA at a specific point called recognition sequence or sites. Each restriction enzyme has its particular restriction site. Thus, recognition sequences play a vital role in giving direction to the restriction enzymes regarding the particular position of cleavage in the DNA.
(iii) Gel electrophoresis is a technique used to separate DNA fragments according to their size. DNA fragments are negatively charged, so they move towards the positive electrode.
Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than larger ones.
When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.
Question. (i) Identify (A) and (B) illustrations in the following :
(ii) Write the term given to (A) and (B) and why ?
(iii) Expand PCR. Mention its importance in biotechnology.
Answer : (i) (A)–AATTC / Sticky end.
(B)–Ori / Origin of Replication.
(ii) Palindromic sequence, because the sequence of base pair reads same on the two strands, when orientation of reading is kept the same.
(iii) PCR – Polymerase Chain Reaction.
Importance – amplification of gene of interest (in vitro).
Question. (a) How do DNA fragments migrate and resolve in Gel electrophoresis?
(b) How lane one is different from lane 2, 3 and 4 in the Gel electrophoresis set up?
(c) How pure DNA fragments are made observable in the visible light?
Answer : (a) The DNA fragments resolve according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
(b) The given agarose gel electrophoresis shows migration of undigested DNA fragments in lane 1 and digested set of DNA fragments in lane 2 to 4.
(c) The separated DNA fragments can be visualized only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
Question. (i) Explain the significance of ‘palindromic nucleotide sequence‘ in the formation of recombinant DNA.
(ii) Write the use of restriction endonuclease in the above process.
Answer : (i) Palindromic nucleotide sequence is the recognition (specific) sequence present both on the vector and on a desired / alien DNA for the action of the same (specific) restriction endonuclease to act upon.
(ii) Same restriction endonuclease binds to both the vector and the foreign DNA, cut each of the two strands of the double helix at specific points in their sugar phosphate backbone of recognition sequence for restriction endonucleases / palindromic sequence of vector and foreign DNA, to cut strand a little away from the centre of the palindrome sites, creates overhanging stretches / sticky ends.
Question. Explain the roles of the following with the help of an example each in recombinant DNA technology:
(a) Restriction Enzymes
Answer : (a) It recognizes a specific sequence of base pairs palindromes and cuts the DNA strand at a specific site.
E.g. EcoRI / Hind II or any other correct example.
(b) Act as vectors / cloning of desired alien gene / foreign gene.
E.g. pBR322 / plasmid of Salmonella / plasmid of Agrobacterium / Ti plasmid/ Tumour inducing plasmid.
Question. Name and explain the technique that helps in the separation of DNA fragments for DNA recombinant technology experiments.
How can these separated DNA fragments be visualised ?
Answer : Gel electrophoresis, Since DNA fragments are negatively charged, they move towards anode (under an electric field) through a medium / matrix / agarose gel. The fragments separate (resolve) according to their size through sieving effect provided by agarose gel. The separated DNA fragments can be visualised after staining the DNA with ethidium bromide, followed by exposure to UV radiation.
Question. (i) Why must bacterial cells be first made ‘competent‘ in r-DNA technology ? How is this process carried out? [KVS]
(ii) Name the method by which an alien DNA can be made to enter (a) plant cell; (b) animal cell.
Answer : (i) Since DNA is hydrophilic, it cannot pass through cell membrane, hence bacterial cells are made competent.
By treatment with a specific concentration of a divalent cations, such as Ca++ which increases efficiency of entry of DNA through the pores of cell wall.
(ii) (a) Plant cells – biolistic / gene guns
(b) Animal cells – Micro injection
Question. Draw a diagram of a typical agarose gel electrophoresis showing migration of undigested and digested sets of DNA fragments. Label
(a) the digested and undigested DNA fragments,
(b) Anode and cathod ends of the plate. Mention the role of electrophoresis in biotechnology.
The cutting of DNA by restiction endonuclease results in fragments of DNA. These fragments can then be separated by (Gel) electrophoresis
Question. Explain the role(s) of the following in Biotechnology :
(i) Restriction endonuclease
(ii) Gel – electrophoresis
(iii) Selectable markers in pBR322.
Answer : (i) Cuts at specific position within the DNA / cuts DNA at specific nucleotide / cuts at palindromic nucleotide sequence.
(ii) Separation of DNA fragments (under the influence of electric field).
(iii) Helps in identifying and eliminating nontransformants from transformants / selection of transformants.
Question. Mention the role of (i) selectable marker, (ii) Ori and (iii) rop in E. coli cloning vector pBR322.
Answer : (i) Selectable marker : Helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.
(ii) Ori : Helps to start replication and any piece of DNA when linked to this sequence can be made to replicate within host cell, responsible for controlling the copy number of the linked DNA.
(iii) Codes for the proteins involved in the replication of the plasmid.
Question. Given below is the diagram of agarose gel kept under UV light :
(i) Mark the positive and negative terminals.
(ii) What is the charge carried by DNA molecule.
(iii) How are the separated DNA fragments finally isolated ?
Answer : (i) Positive terminal-‘B’
(ii) DNA being negatively charged, moves towards the positive electrode (anode).
(iii) By elution-separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.
Question. Rajesh was doing gel electrophoresis to purify DNA fragments. Given below is the sketch of the observations of the experiment performed by him.
(i) At which end he would have loaded the samples and where ?
(ii) Analyse the reason for different positions taken up by the DNA bands.
(iii) Elaborate the step he would have followed to visualize DNA bands.
Answer : (i) He would have loaded the samples near end A, in the wells.
(ii) The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller is the fragment size, the farther it moves.
(iii) After staining the DNA with ethidium bromide followed by exposure to UV radiations, the DNA bands fluoresce.
Long Answer Type Questions
Question. (i) Describe the characteristics a cloning vector must possess.
(ii) Why DNA cannot pass through the cell membrane ? Explain. How is a bacterial cell made ‘competent’ to take up recombinant DNA from the medium ?
Answer : (i) (a) Should have ori / origin of replication, Has selectable marker, genes encoding for an antibiotic resistance / genes encoding for β-galactosidase (b) Has cloning site / recognition site, for the restriction enzyme to recognise.
(ii) DNA is a hydrophilic molecule. Bacterial cell is made competent by treating with specific concentration of Ca++ ions / divalent ions, incubating them on ice, heat shock for a short period and placing it back once again.
Question. Unless the vector and source DNA are cut, fragments separated and joined, the desired recombinant vector molecule cannot be created.
(i) How are the desirable DNA sequence cut ?
(ii) Explain the technique used to separate the cut fragments.
(iii) How are the resultant fragments joined to the vector DNA molecule?
Answer : (i) DNA sequences of the vector as well as the source are cut by the same restriction enzyme like EcoRI, in a palindromic sequence.
(The cut ends overhang as sticky ends in the medium.)
(ii) These cut ends fragments are to be extracted from the culture medium using gel electrophoresis.
This has an agarose gel matrix. Fragments are fed in the wells. DNA are negatively charged so, they move towards anode under an electric field through the gel. Smaller fragments move faster, thus separated.
(iii) Fragments are now added to the medium containing the vector DNA.
The sticky ends facilitates the action of the enzyme ligase and join the source DNA to the vector.
Process of Recombinant DNA Technology
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.
Question. PCR requires very high temperature conditions where most of the enzymes get denatured. How was this problem resolved in a PCR ?
Answer : The thermostable DNA polymerase is used which is isolated from a bacterium called Thermus aquaticus. It remains active during the high temperature this enzyme does not induce denaturation of DNA. This enzymes, taq polymerase carries out amplification of DNA at high temperature.
Question. Write the function of a bioreactor.
Answer : Bioreactors are required to produce large volumes (100 – 1000 litres) of recombinant proteins / desired protein / enzymes.
Question. Name the source of the DNA polymerase used in PCR technique. Mention why it is used ?
Answer : Thermus aquaticus, is the source of DNA polymerase because it is a heat-stable DNA polymerase.
Short Answer Type Questions – l
Question. (i) A recombinant vector with a gene of interest inserted within the gene of α-galactosidase enzyme, is introduced into a bacterium. Explain the method that would help in selection of recombinant colonies from non-recombinant ones.
(ii) Why is this method of selection referred to as “insertional inactivation” ?
Answer : (i) Bacteria are grown in a medium with chromogenic substrate, colonies formed show blue colour-no recombinants, no blue colour – presence of recombinants.
(ii) Gene for the enzyme is inactivated by insertion.
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.
Question. Name two commonly used bioreactors. State the importance of using bioreactors.
Answer : The two types of commonly used bioreactors are:
(i) Stirred tank bioreactor and (ii) Sparged stirred tank bioreactors.
A bioreactor also called fermenter is a specialized container or a vessel required for the production of a number of industrial products such as antibiotics, enzymes and vitamins with the help of microorganisms and microbial reactions going on inside the fermenter. In the bioreactor, a huge amount of raw material or substrates are biologically converted into specific industrial products like vitamins, enzymes, antibiotics, etc. on the industrial scale.
Question. How can bacterial DNA be released from the bacterial cell for biotechnology experiments ?
Answer : DNA is enclosed within the membranes; so, we have to break and open the cell to release DNA along with other macromolecules such as RNA,
proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cell / plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cell), chitinase (fungus).
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 the β-galactosidase) colonies which do not produce colour.
Antibiotic resistance method requires duplicate plating / cumbersome procedure
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.
Short Answer Type Questions – ll
Question. (i) Why was a bacterium used in the first instance of the construction of an artificial recombinant DNA molecule?
(ii) Name the scientists who accomplished this and how?
Answer : (i) Bacterium has a plasmid in which the desired gene is introduced / the gene to be transferred is from a bacterium. The anti-biotic resistance gene / the host cell (bacterial cell) is required for gene cloning / bacteria produce restriction endonucleases.
(ii) Herbert Boyer and Stanley Cohen Antibiotic resistant gene was isolated using restriction enzyme and introduced into the plasmid of bacterium Salmonella typhimurium.
Later the recombinant plasmid was introduced into the bacterium E. coli. so that it could make copies of gene.
Question. (i) List the three steps involved in Polymerase Chain Reaction (PCR).
(ii) Name the source organism of Taq polymerase.
Explain the specific role of this enyme in PCR.
Answer : (i) (a) Denaturation (b) Annealing (c) Extension.
(ii) Thermus aquaticus, it remains active during the high temperature, (induced to denature double stranded DNA) and catalyses polymerisation of DNA.
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.
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. Explain three basic steps to be followed during genetic modification of an organism.
Answer : (i) Identification of DNA with desirable genes, so that the genetically modified organism has largely desirable genes.
(ii) Introduction of the DNA with desirable genes, into the host using vector.
(iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny through cloning.
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.
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.
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.
Question. A schematic representation of polymerase chain reaction (PCR) upto extension stage is given below
Answer the questions that follows :
(i) Name the process ‘a’
(ii) Identify ‘b’
(iii) Identify ‘c’ and mention its importance in PCR.
Answer : (i) Denaturation process.
(iii) Taq DNA polymerase.
Taq polymerase is a thermostable enzyme isolated from a thermophilic bacterium Thermus aquaticus. This enzyme remains functional during high temperature required for extension of DNA
Question. How does β-galactosidase coding sequence act as a selectable marker ? Explain. Why it is a preferred selectable marker to antibiotic resistant genes ?
Answer : (i) Presence of a chromogenic substrate gives blue colour, if the plasmid in the bacteria does not have an insert (non-recombinants).
(ii) With the insert-do not produce any colour, recombinant colonies.
(iii) Selection of recombinants due to inactivation of antibiotics, requires simultaneous plating on two plates having different antibiotics / process is more cumbersome.
Long Answer Type Questions
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.