VBQs Class 12 Biology Biotechnology Principles and Processes

Please refer to VBQs for Class 12 Biology Biotechnology Principles and Processes. All value based questions for Biology Class 12 have been provided with solutions. We have provided below important values questions and answers. Students should learn these solved VBQs for Class 12 Biology as these will help them to gain more marks and help improve understanding of important topics.

Biotechnology Principles and Processes VBQs Class 12 Biology with Answers

Principles of Biotechnology

Very Short Answer Type Questions

Question. Write the two components of the first artificial recombinant DNA molecule constructed by Cohen and Boyer. 
Answer.Two components of first artificial recombinant DNA molecule constructed are : An antibiotic resistance gene and plasmid of Salmonella typhimurium.

Short Answer Type Questions

Question. Explain the work carried out by Cohen and Boyer that contributed immensely in biotechnology.
Answer. Cohen and Boyer contributed to the field of biotechnology by constructing the first recombinant DNA molecule in 1972. They cut the piece of DNA from a plasmid carrying antibiotic resistance gene, using restriction enzymes. This piece of foreign DNA, was linked with the plasmid DNA, acting as a vector with the help of enzyme DNA ligase. This newly formed DNA molecule is called recombinant DNA.

Tools of Recombinant DNA Technology

Very Short Answer Type Questions

Question. Mention the type of host cells suitable for the gene guns to introduce an alien DNA.
Answer. Undifferentiated plant cells are the most suitable host cells for the gene gun to introduce an alien DNA. It is because plant cells have rigid cell wall which can be easily broken down by bombarding them with high velocity micro-particles of gold or tungsten coated with DNA.

Question. Name the technique used for separating DNA fragments in the laboratory.
Answer.Separation of DNA fragments can be done by a technique called agarose gel electrophoresis. In this technique the DNA molecules are separated according to their size, under the influence of an electric field (DNA being negatively charged moves from cathode to anode).

Question. Name the host cells in which micro-injection technique is used to introduce an alien DNA.
Answer.Microinjection technique is used to introduce an alien DNA directly into the nucleus of the animal host cells such as oocytes, eggs and embryo.

Question. Suggest a technique to a researcher who needs to separate fragments of DNA. 
Answer.Separation of DNA fragments can be done by a technique called agarose gel electrophoresis. In this technique the DNA molecules are separated according to their size, under the influence of an electric field (DNA being negatively charged moves from cathode to anode).

Question. What is the role of ethidium bromide during agarose-gel electrophoresis of DNA fragments?
Answer. DNA fragments can be seen only after staining. Ethidium bromide is used to stain DNA fragments followed by exposure to UV radiation. This gives bright orange colour to DNA fragments which helps to view separated DNA fragments.

Question. State what happens when an alien gene is ligated at SalI site of pBR322 plasmid.
Answer.When an alien gene is ligated at SalI site of pBR322, the gene tetR becomes non functional and plasmid loses its tetracycline resistance. Hence, the cell possessing such recombinant pBR322 will not be able to grow on tetracycline.

Question. Why is it essential to have a ‘selectable marker’ in a cloning vector? 
Answer. Selectable marker helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. Hence, they are considered as essential property in a cloning vector.

Question. How can retroviruses be used eficiently in biotechnology experiments inspite of them being disease causing? 
Answer.Retroviruses are first disarmed (disease causing gene is removed/inactivated), this disarmed virus do not cause disease and hence are used to transfer desirable genes into host cells. So, inspite of them being disease causing, it (disarmed retrovirus) is used eficiently in biotechnology experiments.

Question. Why is ‘plasmid’ an important tool in biotechnology experiments? 
Answer. Plasmid have the ability to replicate within bacterial cells independent of the control of chromosomal DNA and have high copying number, therefore any alien DNA ligated to it, also multiplies to equal the copy number of plasmids. So, it is used as a vector in gene cloning experiments and thus plays a role of an important tool in biotechnology.

Question. Mention the use of cloning vector in biotechnology. 
Answer. Cloning vectors are essential in biotechnological experiment as they help in transferring a fragment of foreign DNA (gene of interest) into suitable host. They help in cloning, have high copy number and help in easy linking of alien DNA.

Question. In the year 1963, two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated. How did the enzymes act to restrict the growth of the bacteriophage?
Answer. In 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA while the other cut DNA at specific sequences i.e., restriction endonuclease. To restrict the growth of bacteriophage, the E. coli recognises and cut foreign DNA into pieces by restriction endonucleases. And in order to prevent cleavage of its own DNA by these enzymes, it modifies its DNA by adding methyl groups. And thus remain unrecognised.

Question. How is the action of exonuclease dierent from that of endonuclease? 
Answer. Differences between action of exonucleases and endonucleases are as follows :

Question. State what happens when an alien gene is ligated at PvuI site of pBR322 plasmid. 
Answer.When an alien gene is ligated at PvuI site of pBR322 plasmid the transformant cell loses ampicillin-resistance as ampR gene becomes nonfunctional.
Thus, the recombinant will be unable to grow in presence of ampicillin.

Question. Why do DNA fragments move towards the anode during gel-electrophoresis?
Answer. DNA is a negatively charged molecule hence during gel electrophoresis it move towards anode (positive electrode) under the influence of electrical field.

Short Answer Type Questions

Question. Write the role of ori and ‘restriction’ site in a cloning vector pBR322. 
Answer. Origin of replication (ori) site in cloning vector pBR322 is a sequence from where replication starts. Any piece of DNA when linked to this sequence can be made to replicate within host cell. Restriction site within the markers tetR and ampR genes permit an easy selection for cells transformed or the recombinant pBR322.

Question. How do ‘ori’ and ‘cloning sites’ facilitate cloning into a vector?
Answer. Ori sites are the origin of replication sites which control replication of the DNA in which they are present. Cloning of a vector containing rDNA requires its multiplication to produce large number of copies and ori is essential for it. Cloning sites are the specific sites in vector that possess recognition sequences for a particular enzyme. It enables insertion of foreign DNA segment into that particular site.

Question. How does a restriction nuclease function? Explain. 
Answer. Restriction nucleases acts as molecular scissors or chemical scalpels. Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its sepcific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones. Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.

Question. What is EcoRI? How does EcoRI differ from an exonuclease? 
Answer. EcoRI is a restriction endonuclease enzyme it recognises base sequences 5′-GAATTC-3′ 3′-CTTAAG-5′ in DNA duplex and cuts each of the two strands between G and A. On the other hand, exonuclease remove nucleotide from the terminal ends of DNA in one strand of duplex. Hence, EcoRI cut each of the two strand of DNA duplex at specic point whereas exonuclease remove nucleotide from the terminal ends (either 5′ or 3′) of DNA in one strand of duplex.

Question.(a) Explain how to find whether an E. coli bacterium has transformed or not when a recombinant DNA bearing ampicillin resistant gene is transferred into it.
(b) What does the ampicillin resistant gene act as in the above case? 
Answer. 
(a) If the recombinant DNA bearing ampcillin resistant is transferred into E.coli bacteria, it will confer resistance to ampicillin and therefore will grow on ampicillin containing medium. However the nontransformants will not grow on same medium. The recombinants or transformed E.coli can be selected by use of selectable markers. The transformants must be plated on a plate bearing ampicillin containing medium. Hence, transformants can be selected from non-transformants.
(b) Ampicillin resistant gene act as selectable marker and helps in selecting the transformant in the above case.

Question. (a) Illustrate the recognition sequence of EcoRI and mention what such sequences are called.
(b) How does restriction endonuclease act on a DNA molecule? 
Answer.(a) Recognition sequence of EcoRI is
5′ – GAATTC-3′
3′ – CTTAG-5′
Such sequences are called palindrome sequence.
(b) Restriction endonuclease recognise the base sequence at palindrome site in DNA duplex and cuts its strands.

Question. How are ‘sticky ends’ formed on a DNA strand? Why are they so called? 
Answer. When restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase.

Question. How is insertional inactivation of an enzyme used as a selectable marker to differentiate recombinants from non-recombinants?
Answer. Insertional inactivation refers to the process where insertion of rDNA within the coding sequence of an enzyme causes its inactivation. The non-recombinants having intact functional gene e.g. b-galactosidase produce blue colour with chromogenic substrate but when rDNA is inserted within the coding sequence of enzyme b-galactosidase, recombinants do not produce any colour. Hence recombinants can be easily differentiated from non-recombinants due to insertional inactivation.

Question. How is DNA isolated in purified form from a bacterial cell? 
Answer.DNA is isolated in purified form from a bacterial cell in following steps :
(i) Break the cell to release DNA along with other macromolecules. This can be done by treating cells with enzymes like lysozyme in case of bacteria, cellulase in case of plant cells and chitinase for fungus.
(ii) RNA and proteins are also present with DNA.These can be removed by ribonuclease and proteases respectively.
(iii) Similarly other molecules (if any) are removed by appropriate treatments.
(iv) Purified DNA can be precipitated out after treating with chilled ethanol and DNA fragments can be seen as fine threads in the suspension.

Question. Explain palindromic nucleotide sequence with the help of a suitable example. 
Answer. The palindrome sequence in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
For example, the following sequences reads the same on the two strands in 5′ → 3′ direction. This is also true if read in the 3′ → 5′ direction.
5′ – GAATTC-3′
3′ – CTTAAG-5′

Question. (a) Mention the difference in the mode of action of exonuclease and endonuclease.
(b) How does restriction endonuclease function?
Answer.(a) Differences between action of exonucleases and endonucleases are as follows :

(b) Restriction nucleases acts as molecular scissors or chemical scalpels. Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its sepcific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones. Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.

Question. (a) How is an exonuclease functionally different from an endonuclease?
(b) Give an example of any two endonucleases other than Sal I. 
Answer.(a) Differences between action of exonucleases and endonucleases are as follows :

(b) Two examples of endonucleases other than SalI are EcoRI and HindIII.

Question. How can DNA segments, separated by gel electrophoresis be visualised and isolated?
Answer. The central dye in agarose gel electrophoresis is ethidium bromide. It has the unique property of fluorescence under UV light when intercalated with DNA. By running DNA through an ethidium bromidetreated gel and exposing it to UV light, distinct bands of DNA become visible. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution.

Question. (a) A recombinant vector with a gene of interest inserted within the gene of b-galactosidase enzyme, is introduced into a bacterium. Explain the method that would help in selection of recombinant colonies from nonrecombinant ones.
(b) Why is this method of selection referred to as “insertional inactivation”?
Answer. 
(a)Insertional inactivation refers to the process where insertion of rDNA within the coding sequence of an enzyme causes its inactivation. The non-recombinants having intact functional gene e.g. b-galactosidase produce blue colour with chromogenic substrate but when rDNA is inserted within the coding sequence of enzyme b-galactosidase, recombinants do not produce any colour. Hence recombinants can be easily differentiated from non-recombinants due to insertional inactivation.
(b) In this method, insertion of recombinat DNA in the coding sequence of enzyme b-galactosidase causes its inactivation hence named insertional inactivation.

Question. List the key tools used in recombinant DNA technology. 
Answer. Biological or key tools used in recombinant DNA technology are :
(i) Enzymes : Different kinds of specific enzymes used in recombinant DNA technology are lysing enzymes (Used to open up the cells to get DNA), it includes lysozyme, cellulose and chitinase and cleaving enzymes (enzymes used to break DNA molecules) it includes exonuclease, endonuclease and restriction endouclease.
(ii) Cloning vectors : These are DNA molecules that can carry foreign DNA segment and replicate inside a host cell. It may be plasmids, a bacteriophage, cosmids, Yeast Artifical Chromosomes(YACs), Bacterial Artifical Chromosomes (BACs) and viruses.
(iii) Competent host : A competent host is essential for transformation with recombinant DNA. It includes DNA mediated or vector mediated gene transfer and direct or vectorless gene transfer (microinjection, electroporation, chemical mediated gene transfer, biolistic method or gene gun method).

Question. Explain the role of Ti plasmids in biotechnology.
Answer.Agrobacterium tumifaciens is a soilinhabiting bacterium that may invade growing plants at the junction of root and stem, where it can cause a cancerous growth known as a crown gall. A. tumifaciens contains Ti plasmid which carries gene for tumour formation. For using Agrobacterium tumifaciens as a cloning vector researchers deleted the genes which governs auxin and cytokinin production (the oncogene) from T-DNA of Ti plasmid. It is known as disarming. After disarming, this T-DNA is inserted into chromosomes of the host plant where it produces copies of itself.

Question. Why is making cells competent essential for biotechnology experiments? List any two ways by which this can be achieved. 
Answer. Competent host is essential for biotechnology experiment. Since DNA is a hydrophilic molecule,it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA i.e., made competent.
This can be achieved by :
(i) treatment of DNA with divalent cation of CaCl₂ or rubidium chloride. Treating them with a specific concentration of a divalent cation, increases the eficiency with which DNA enters the bacterium through pores in its cell wall.
(ii) Heat shock treatment of DNA. Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock) and then putting them back on ice.This enables the bacteria to take up the recombinant DNA.

Question. (a) A recombinant DNA is formed when sticky ends of vector DNA and foreign DNA join.
(b) Explain how the sticky ends are formed and get joined. 
Answer.(a) When restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase.
(b) The sticky ends get joined end to end with the help of enzyme DNA ligase.

Question. State the role of UV-light and ethidium bromide during gel electrophoresis of DNA fragments.
Answer.DNA fragments can be seen only after staining. Ethidium bromide is used to stain DNA fragments followed by exposure to UV radiation. This gives bright orange colour to DNA fragments which helps to view separated DNA fragments.

Question. A and B are the two different cloning vectors in two different bacterial colonies cultured in chromogenic substrate. Bacterial colonies with cloning vector A were colourless whereas those with B were blue coloured. Explain giving reasons the cause of the difference in colour that appeared. 
Answer. Presence of the insert within a gene results in insertional inactivation of the enzyme b-galactosidase, hence bacterial colonies do not produce any colour. Therefore, bacterial colonies with cloning vector A are colourless as they are recombinants with the insert and bacterial colonies with cloning vector B are blue coloured as they are non-recombinants.

Question. Write any four ways used to introduce a desired DNA segment into a bacterial cell in recombinant technology experiments.
Answer.Four ways to form a desired DNA segment into a host cell are (i) Electroporation, (ii) Chemical mediated gene transfer (iii) Microinjection and (iv) biolistic gun.

Question. Name the natural source of agarose. Mention one role of agarose in biotechnology.
Answer.Agarose is a commonly used as matrix in agarose gel electrophoresis. It is extracted from sea weeds. In recombinant DNA technology, agarose is used to separate DNA fragments according to their size.

Question.

Study the linking of DNA fragments shown above.
(a) Name ‘A’ DNA and ‘B’ DNA.
(b) Name the restriction enzyme that recognises this palindrome.
(c) Name the enzyme that can link these two DNA fragments.
Answer.(a) A = Vector DNA
B = Foreign DNA
(b) Restriction enzyme EcoRI recognises this palindrome.
(c) DNA ligase link these two DNA fragments.

Question. What is a plasmid? 
Answer.Plasmids are extra-chromosomal, self replicating, usually circular, double stranded DNA molecules, found naturally in many bacteria and also in some yeast.

Short Answer Type Questions

Question. (a) Explain the basis on which the gel electrophoresis technique works.
(b) Write any two ways the products obtained through this technique can be utilised.
Answer.(a) After the cutting of DNA by restriction enzyme, fragments of DNA are formed. Separation of DNA fragments according to their size or length is done by a technique called agarose gel electrophoresis. It is a technique of separation of molecules such as DNA, RNA or protein, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, viz., positively charged molecules move towards cathode (–ve electrode) and negatively charged molecules travel towards anode (+ve electrode) through a medium/ matrix. Most commonly used matrix is agarose. DNA fragments separate according to size through the pores of agarose gel. Hence the smaller, the fragment size, the farther it moves.
The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr) followed by exposure to UV radiation. The fragments are visible as bright orange coloured bands.
(b) The product obtained can be used in the following way :
(i) Fragments of DNA obtained can be used to construct a recombinant DNA molecule by joining them with cloning vector
(ii) The desired DNA fragment can be amplified using polymerase chain reaction (PCR).

Question. Name and describe the technique that helps in separating the DNA fragments formed by the use of restriction endonuclease.
Answer. After the cutting of DNA by restriction enzyme, fragments of DNA are formed. Separation of DNA fragments according to their size or length is done by a technique called agarose gel electrophoresis. It is a technique of separation of molecules such as DNA, RNA or protein, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, viz., positively charged molecules move towards cathode (–ve electrode) and negatively charged molecules travel towards anode (+ve electrode) through a medium/ matrix. Most commonly used matrix is agarose. DNA fragments separate according to size through the pores of agarose gel. Hence the smaller, the fragment size, the farther it moves.
The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr) followed by exposure to UV radiation. The fragments are visible as bright orange coloured bands.

Question. State the functions of the following in the cloning vector pBR322 :
(a) Ori,
(b) rop, and
(c) HindIII sites
Answer.(a) Origin of replication (Ori) is a specific sequence of DNA bases which is responsible for initiating replication. It is also responsible for controlling the copy number of the linked DNA.
(b) rop in pBR322 encodes for a protein involved in replication of plasmid.
(c) HindIII is restriction site in pBR322. It is the site where HindIII endonucleaese make a cut so that a foreign DNA segment can be introduced to this vector.

Question. (a) Name the selectable markers in the cloning vector pBR322? Mention the role they play.
(b) Why is the coding sequence of an enzyme β-galactosidase a preferred selectable marker in comparison to the ones named above? 
Answer. 
(a) Plasmid pBR322 is a most widely used cloning vector. It has two resistance genes ampicillin resistance (ampR) and tetracycline resistance (tetR) which are considered useful as selectable markers. Selectable markers help in identifying and eliminating non-transformants and selectively permitting the growth of transformants. Bacterial cells containing recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline.
(b) Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, b-galactosidase. This results into inactivation of the enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the b-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

Question. (a) Draw schematic diagrams of segments of a vector and a foreign DNA with the sequence of nucleotides recognised by EcoRI.
(b) Draw the vector DNA segment and foreign DNA segments after the action of EcoRI and label the sticky end produced.
Answer.

Fig: Segments of a vector and a foreign DNA with the sequence of nucleotide recognised by EcoRI.

Fig: Vector DNA segment and foreign DNA segment after the action of EcoRI.

Question. DNA being hydrophilic cannot pass through the cell membranes of a host cell. Explain how does recombinant DNA get introduced into the host cell to transform the latter. 
Answer.Competent host is essential for biotechnology experiment. Since DNA is a hydrophilic molecule,it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA i.e., made competent.
This can be achieved by :
(i) treatment of DNA with divalent cation of CaCl₂ or rubidium chloride. Treating them with a specific concentration of a divalent cation, increases the eficiency with which DNA enters the bacterium through pores in its cell wall.
(ii) Heat shock treatment of DNA. Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock) and then putting them back on ice.This enables the bacteria to take up the recombinant DNA.

Question. Draw a schematic sketch of pBR322 plasmid and label the following in it :
(a) Any two restriction sites.
(b) Ori and rop genes.
(c) An antibiotic resistant gene.
Answer.

Question. (a) Why must a cell be made ‘competent’ in biotechnology experiments? How does calcium ion help in doing so?
(b) State the role of ‘biolistic gun’ in biotechnology experiments.
Answer. (a)Competent host is essential for biotechnology experiment. Since DNA is a hydrophilic molecule,it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA i.e., made competent.
This can be achieved by :
(i) treatment of DNA with divalent cation of CaCl₂ or rubidium chloride. Treating them with a specific concentration of a divalent cation, increases the eficiency with which DNA enters the bacterium through pores in its cell wall.
(ii) Heat shock treatment of DNA. Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock) and then putting them back on ice.This enables the bacteria to take up the recombinant DNA.
(b) Biolistic gun helps in the process of gene transfer into the host cell without using a vector. In biolistic method or gene gun method, tungsten or gold particles, coated with foreign DNA are bombarded into target cells at a very high velocity. This method is suitable for plants, but is also used to insert genes into animal that promote tissue repair into cells (particularly cancer of mouth) near wounds. It has made great impact in the field of vaccine development.

Question. (a) Why are restriction endonucleases so called?
(b) What is a palindromic nucleotide sequence? How do restriction endonucleases act on palindromic sites to create ‘sticky ends’?
Answer.(a) Restriction endonucleases are so called because they cut DNA duplex at specific points.
Their single stranded free ends are called ‘sticky ends’ which can be joined end to end by DNA ligases.
(b)The palindrome sequence in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
For example, the following sequences reads the same on the two strands in 5′ → 3′ direction. This is also true if read in the 3′ → 5′ direction.
5′ – GAATTC-3′
3′ – CTTAAG-5′
When restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase.

Question. (a) Name the organism in which the vector shown is inserted to get the copies of the desired gene.
(b) Mention the area labelled in the vector responsbile for controlling the copy number of the inserted gene.
(c) Name and explain the role of a selectable marker in the vector shown.
Biotechnology : Principles and Processes

Answer.(a) The vector shown is inserted into Escherichia coli to get the copies of the desired gene.
(b) Origin of replication (ori) is a sequence from where replication starts and is also responsible for controlling the copy number of the linked DNA.
(c) The sequence ampR and tetR are considered useful selectable markers. The given vector shows ampR selectable marker. Vector requires a selectable marker, because it helps in identifying and eliminating non – transformants and selectively permitting the growth of the transformants.
(a) If the recombinant DNA bearing ampcillin resistant is transferred into E.coli bacteria, it will confer resistance to ampicillin and therefore will grow on ampicillin containing medium. However the nontransformants will not grow on same medium. The recombinants or transformed E.coli can be selected by use of selectable markers. The transformants must be plated on a plate bearing ampicillin containing medium. Hence, transformants can be selected from non-transformants.
(b) Ampicillin resistant gene act as selectable marker and helps in selecting the transformant in the above case.

Question. (a) EcoRI is a restriction endonuclease. How is it named so? Explain.
(b) Write the sequence of DNA bases that the enzyme recognises. Mention the point at which the enzyme makes a cut in the DNA segment.
Answer. 
(a) Enzyme EcoRI is named as follows : The capital letter E comes from the genus Escherichia : The letters co are derived from the species name coli. The letter R is from RYI3 (strain). The Roman number I indicates that it was the first enzyme isolated from the bacterium E. coli RYI3.
(b) EcoRI is a restriction endonuclease enzyme it recognises base sequences 5′-GAATTC-3′ 3′-CTTAAG-5′ in DNA duplex and cuts each of the two strands between G and A. On the other hand, exonuclease remove nucleotide from the terminal ends of DNA in one strand of duplex. Hence, EcoRI cut each of the two strand of DNA duplex at specic point whereas exonuclease remove nucleotide from the terminal ends (either 5′ or 3′) of DNA in one strand of duplex.

Question. Read the following base sequence of a certain DNA strand and answer the questions that follow :

(a) What is called a ‘palindromic sequence’ in a DNA?
(b) Write the palindromic nucleotide sequence shown in the DNA strand given and mention the enzyme that will recognise such a sequence.
(c) State the significance of enzyme that identify palindromic nucleotide sequences.
Answer.(a) The palindrome sequence in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
For example, the following sequences reads the same on the two strands in 5′ → 3′ direction. This is also true if read in the 3′ → 5′ direction.
5′ – GAATTC-3′
3′ – CTTAAG-5′
(b) e palindrome sequence shown in the given DNA strand is
5′-GAATTC-3′
3′-CTTAAG-5′
This is the recognition sequence for restriction enzyme EcoRI.
(c) Restriction endonuclease such as EcoRI, HindIII, Bam HI acts as molecular scissors or chemical scalpels. They serve as the tools for cutting DNA molecules at specific palindromic sites, which is the basic requirement for gene cloning or recombinant DNA technology.

Question. EcoRI is used to cut a segment of foreign DNA and that of a vector DNA to form a recombinant DNA. Show with the help of schematic diagrams.
(a) The set of palindromic nucleotide sequence of base pairs the EcoRI will recognise in both the DNA segments. Mark the site at which EcoRI will act and cut both the segments.
(b) Sticky ends of formed on both the segments where the two DNA segments will join later to form a recombinant DNA.
Answer.

Fig: Segments of a vector and a foreign DNA with the sequence of nucleotide recognised by EcoRI.

Question. Explain any three methods to force ‘alien’ or recombinant DNA into host cells
Answer.The three methods which force ‘alien’ or recombinant DNA into host cells are: electroporation,biolistic method and microinjection.
(i) Electroporation : In this method, electrical impulses induce transient pores in the plant cell membrane through which foreign DNA molecules are incorporated into cells.
(ii) Biolistic Method : Biolistic is a means of introducing DNA into cells that involves bombardment of cells with high-velocity microprojectiles coated with DNA. In biolistic method tungsten or gold particles, coated with foreign DNA are bombarded into target cells at a very high velocity.
(iii) Microinjection : It is the introduction of foreign gene into plant cell or animal cell by using microneedles or micropipettes.

Question. (a) Identify the selectable markers in the diagram of E. coli vector shown below.

(b) How is the coding sequence of b-galactosidase considered a better marker than the ones identied by you in the diagram?
Explain. 
Answer.(a) Selectable marker in given cloning vector pBR322 are ampicillin resistance gene(a), and tetracycline resistance gene(d). ey help in selecting transformant from non-transformant ones.
(b) Biolistic gun helps in the process of gene transfer into the host cell without using a vector. In biolistic method or gene gun method, tungsten or gold particles, coated with foreign DNA are bombarded into target cells at a very high velocity. This method is suitable for plants, but is also used to insert genes into animal that promote tissue repair into cells (particularly cancer of mouth) near wounds. It has made great impact in the field of vaccine development.

Question. Name and explain the technique used in the separation and isolation of DNA fragments to be used in recombinant DNA technology.
Answer. After the cutting of DNA by restriction enzyme, fragments of DNA are formed. Separation of DNA fragments according to their size or length is done by a technique called agarose gel electrophoresis. It is a technique of separation of molecules such as DNA, RNA or protein, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, viz., positively charged molecules move towards cathode (–ve electrode) and negatively charged molecules travel towards anode (+ve electrode) through a medium/ matrix. Most commonly used matrix is agarose. DNA fragments separate according to size through the pores of agarose gel. Hence the smaller, the fragment size, the farther it moves.
The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr) followed by exposure to UV radiation. The fragments are visible as bright orange coloured bands.

Question. (a) What is EcoRI? What does ‘R’ represent in this?
(b) Give the palindromic nucleotide sequence recognised by it.
(c) Explain its action.
Answer.(a) Enzyme EcoRI is named as follows : The capital letter E comes from the genus Escherichia : The letters co are derived from the species name coli. The letter R is from RYI3 (strain). The Roman number I indicates that it was the first enzyme isolated from the bacterium E. coli RYI3.
(b) EcoRI is a restriction endonuclease enzyme it recognises base sequences 5′-GAATTC-3′ 3′-CTTAAG-5′ in DNA duplex and cuts each of the two strands between G and A. On the other hand, exonuclease remove nucleotide from the terminal ends of DNA in one strand of duplex. Hence, EcoRI cut each of the two strand of DNA duplex at specific point whereas exonuclease remove nucleotide from the terminal ends (either 5′ or 3′) of DNA in one strand of duplex.

Question. Why are genes encoding resistance to antibiotics considered useful selectable markers for E. coli cloning vector? Explain with the help of one example. 
Answer. Genes encoding resistance to antibiotics are considered useful selectable markers for E. coli cloning vector because they help in selecting transformant cell from non-transformant ones. The genes encoding resistance to antibiotics such as tetracycline, ampicillin, kanamycin or chloramphenicol etc. are useful selectable markers for E. coli. The common E. coli cells are not resistant to any of these antibiotics. Plasmid pBR322 has two antibiotic resistance genes – ampicillin resistance (ampR) and tetracycline resistance (tetR) which are considered useful for selectable markers.
The presence of restriction sites within the markers tetR and ampR permits an easy selection for cells transformed with the recombinant pBR322. For example, insertion of the DNA fragment into the plasmid using enzyme PstI or PvuI places the DNA insert within the gene ampR. This makes ampR nonfunctional. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline.

Question. (a) Explain the role of restriction endonucleases in recombinant DNA technology.
(b) Name the endonuclease that was first discovered. 
Answer.(a) Restriction endonuclease such as EcoRI, HindIII, Bam HI acts as molecular scissors or chemical scalpels. They serve as the tools for cutting DNA molecules at specific palindromic sites, which is the basic requirement for gene cloning or recombinant DNA technology.
(b) The first discovered restriction endonuclease was HindII.

Question. (a) Write the palindromic nucleotide for the following DNA segment : 5′-GAATTC-3′
(b) Name the restriction endonuclease that recognises this sequence.
(c) How are ‘sticky-ends’ produced? Mention their role. 
Answer.(a) Palindromic nucleotide sequence for the
given DNA segment is : 3′ CTTAAG-5′
(b) EcoRI is a restriction endonuclease enzyme it recognises base sequences 5′-GAATTC-3′ 3′-CTTAAG-5′ in DNA duplex and cuts each of the two strands between G and A. On the other hand, exonuclease remove nucleotide from the terminal ends of DNA in one strand of duplex. Hence, EcoRI cut each of the two strand of DNA duplex at specic point whereas exonuclease remove nucleotide from the terminal ends (either 5′ or 3′) of DNA in one strand of duplex.
(c) When restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase. 

Question. Why is Agrobacterium tumefaciens a good cloning vector? Explain. 
Answer.Agrobacterium tumifacines is considered as a good cloning vector because it is used for introducing genes of desirable traits into plants. Properties of Ti plasmid of Agrobacterium are as follows :
(i) They are self replicating.
(ii) They possess sites for insertion of foreign gene which needs to be introduced.
(iii) They possess selectable markers.

Question. Draw pBR322 cloning vector. Label ‘ori’, ‘rop’ and any one antibiotic resistance site on it and state their functions. 
Answer.

Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.
rop : rop codes for protein involved in the replication of plasmid ampR: gene for ampicillin resistance which help in selecting transformants.

Long Answer Type Questions

Question. Name the enzymes that are used for the isolation of DNA from bacterial and fungal cells for recombinant DNA technology.
Answer.In recombinant DNA technology, enzymes that are used for isolation of DNA from bacterial and fungal cells are lysozyme and chitinase respectively.

Question. (a) With the help of diagrams show the different steps in the formation of recombinant DNA by action of restriction endonuclease enzyme EcoRI.
(b) Name the technique that is used for separating the fragments of DNA cut by restriction endonucleases. 
Answer.

Competent host is essential for biotechnology experiment. Since DNA is a hydrophilic molecule,it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA i.e., made competent.
This can be achieved by :
(i) treatment of DNA with divalent cation of CaCl₂ or rubidium chloride. Treating them with a specific concentration of a divalent cation, increases the eficiency with which DNA enters the bacterium through pores in its cell wall.
(ii) Heat shock treatment of DNA. Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock) and then putting them back on ice.This enables the bacteria to take up the recombinant DNA.

Question. (a) Describe the characteristics a cloning vector must possess.
(b) Why DNA cannot pass-through the cell membrane? Explain. How is bacterial cell made ‘competent’ to take up recombinant DNA from the medium? 
Answer.(a) A cloning vector must possess the following characterstics:
(i) Origin of replication (Ori) : Ori is a sequence from where replication starts and is also responsible for controlling the copy number of the linked DNA.
(ii) Selectable marker : Selectable markers help in selecting transformant host cell from nontransformant ones.
(iii) Cloning sites : A vector must have unique recognition site to link foreign DNA. Presence of a particular cloning/recognition site enables the particular enzyme to cut the vector DNA.

Question. Unless the vector and source DNA are cut, fragments separated and joined, the desired recombinant vector molecule cannot be created.
(a) How are the desirable DNA sequences cut?
(b) Explain the technique used to separate the cut fragments.
(c) How are the resultant fragments joined to the vector DNA molecule? 
Answer. (a) Desirable DNA sequences are cut by the use of enzyme restriction endonuclease.
When restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase.
(b) After the cutting of DNA by restriction enzyme, fragments of DNA are formed. Separation of DNA fragments according to their size or length is done by a technique called agarose gel electrophoresis. It is a technique of separation of molecules such as DNA, RNA or protein, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, viz., positively charged molecules move towards cathode (–ve electrode) and negatively charged molecules travel towards anode (+ve electrode) through a medium/ matrix. Most commonly used matrix is agarose. DNA fragments separate according to size through the pores of agarose gel. Hence the smaller, the fragment size, the farther it moves.
The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr) followed by exposure to UV radiation. The fragments are visible as bright orange coloured bands.
(c) DNA ligase help to join resultant fragments to the vector DNA molecule.DNA ligases join two individual fragments of double stranded DNA by the formation of phosphodiester bond between them.