Question. Write the two components of first artificial recombinant DNA molecule constructed by Cohen and Boyer.
Answer. The two components of first artificial recombinant DNA molecule constructed by Cohen and Boyer are:
(i) antibiotic resistance gene
(ii) plasmid of Salmonella typhimurium

Question. Write any four ways used to introduce a desired DNA segment into a bacterial cell in recombinant technology experiments.
Answer. Ways to introduce desired DNA into bacterial cell are: (i) microinjection (ii) disarmed pathogen vectors
(iii) portion by bivalent cation such as calcium
(iv) biolistic or gene gun

Question. Mention the uses of cloning vector in biotechnology?
Answer. Uses of cloning vector in biotechnology.
(i) Helps in linking the foreign/alien DNA with that of host’s DNA.
(ii) Help in the selection erf recombinants from the non-recombinants.

Question. Biotechnologists refer to Agrobacterium tumefaciens as natural genetic engineer of plants. Give reasons to support the statement.
Answer. Agrobacterium tumefaciens is a pathogen of several dicot plants. It is used as a natural genetic engineer because it is able to deliver a piece of its DNA (called T-DNA) to transform normal plant cells into tumour cells and direct the tumour cells to synthesise the chemicals required by the pathogen.

Question. Why do DNA fragments move towards the anode during gel electrophoresis?
Answer. DNA fragments are negatively charged molecules and hence, moves toward the anode during gel electrophoresis. Cut DNA at specific points.

Question. How is the action of exonuclease different from that of endonuclease.
Answer. Exonuclease removes nucleotides from the ends of DNA, while endonuclease cuts the DNA at specific positions.

Question. What is the role of ethidium bromide during agarose gel electrophoresis of DNA fragments?
Answer. The separated DNA fragments during agarose gel electrophoresis are visualised after staining the DNA with ethidium bromide, in UV light. This staining imparts DNA a bright orange colour.

Question. Why is the enzyme cellulase used for isolating genetic material from plant cells but not for animal cells?
Answer. Cellulase is used for digesting the cellulosic cell wall of plant cells. Animal cells do not contain cell wall, so cellulase is not required.

2 MARK QUESTIONS

Question. Write the role of Ori and restriction site in a cloning vector pBR322.
Answer. Ori is a sequence of DNA from where replication starts. Any piece of DNA that needs to replicate in the host cell has to be linked to it. Cloning sites refers to the site/sequence of DNA where the alien DNA is linked.

Question. How are sticky ends formed on a DNA strand? Why are they so called?
Answer. Sticky ends on DNA are formed by action of enzymes restriction endonucleases. These enzymes cut the strand of DNA a little away from the centre of the palindrome sequence between the same two bases on both the strands. This results in single stranded stretches on both the complementary strands at their ends. These overhanging stretches are called sticky ends as they form hydrogen bonds with the complementary base pair sequences.

Question. How is insertional inactivation of an enzyme used as a selectable marker to differentiate recombinants from non-recombinants?
Answer. The insertional activation of J3-galactosidase enzyme, i.e. by inserting the desired gene in the coding region of enzyme, results in inactivation of (3-galactosidase gene in recombinants. The recombinant on transformed hosts are unable to produce any colour when grown on chromogenic substrate, thus acting as a selectable marker to differentiate recombinants from non-recombinants.

Question. Why is making cells competent essential for biotechnology experiments? List any two ways by which this can be achieved.
Answer. Since, DNA molecules are hydrophilic, they cannot pass through cell membranes. For recombinant DNA to be integrated into vector or host genome it is necessary for the DNA to be inserted in the cell. Therefore, making the host cells competent is necessary in biotechnology experiments. The two ways by which cells can be made competent to take up DNA are: (i) Chemical action – By increasing concentration of divalent cation, calcium, thereby increasing the efficiency of DNA entering through pores in cell, wall. (ii) Heat shock treatment – Incubating the cells with recombinant DNA on ice, followed by brief treatment of heat at 42 °C and again putting them back on ice.

Question. (i) A recombinant vector with a gene of interest inserted within the gene of a-galactosidase enzyme is introduced into a’ bacterium. Explain the method that would help in selection of recombinant colonies from non-recombinant colonies. (ii) Why is this method of selection referred to as insertional inactivation?
Answer. (i) The recombinant colonies can be differentiated from non-recombinant colonies by their inability to produce colour in the presence of a chromogenic substrate. The recombinants do not produce any colour while, the non-recombinants produce a blue colour with chromogenic substrate in the medium. (ii) The enzyme a-galactosidase become inactivated on insertion of recombinant DNA, within the coding sequence of enzyme. Thus, the method is called insertional inactivation.

Question. Explain the role of Ti plasmids in biotechnology.
Answer. The Ti plasmid of Agrobacterium is responsible for the natural transformation of plant cells into tumours. So, it is modified into a non-pathogen ic vector but still is able to deliver the DNA. This disarmed plasmid of Agrobacterium is used as a vector for the transformation of plant cells, thus proved to play an important role in biotechnology.

Question. Study the diagram given below and answer the following questions 

(i) Why have DNA fragments in bank D moved farther away in comparison to those in band C?
(ii) Identify the anode end in diagram.
(iii) How are these DNA fragments visualised
Answer. (i) In band D, DNA fragments are smaller than those on band C. The fragments separate according to their size through the sieving effect provided by the gel So, the smaller fragments move farther away than the larger ones.
(ii) B is anode end in the diagram.
(iii) Gel containing DNA fragments is stained with ethidium bromide and exposed to UV radiation. Orange colour bands of DNA becomes visible.

Question. How are the DNA fragments separated by gel electrophoresis visualised and separated for use in constructing recombinant DNA?
Answer. The separated DNA fragments are stained with ethidium bromide. (i) By the exposure to UV radiation, the separated DNA fragments become visible as orange-coloured bands.
(ii) The separated bands of DNA are cut out from the agarose gel and DNA is extracted from these gel pieces, this process is called elution.

Question. (i) Illustrate the recognition sequence of Eco RI and mention what such sequences are called?
(ii) How does restriction endonuclease act on a DNA molecule?
Answer.(i) Recognition sequence of Eco Rl 5′- G A A T T C -3′ 3′- C T T A A G -3′ These sequences are called palindromic nucleotide sequences.
(ii) Restriction endonuclease acts on specified length of a DNA and binds to the DNA at the specific recognition sequence. It cuts both the double strands of DNA, at the sugar phosphate backbones, a little away from the centre of palindromic sites in between

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

Answer. Figure is a simple stirred-tank bioreactor. Bioreactors are used to produce large quantities of the desired gene products the specific sequence or points.

3 MARK QUESTIONS

Question. Name and describe the technique that helps in separating the DNA fragments formed by the use of restriction endonuclease. 
Answer. DNA fragments formed by the use of restriction endonucleases are separated by gel electrophoresis.
(i) DNA fragments are negatively charged molecules. Thus, they move towards the anode under electric field through the medium.
(ii) DNA fragments separate according to their size due to sieving effect of agarose gel.
(iii) The separated DNA fragments can be viewed by staining the DNA with ethidium bromide followed by exposure to UV radiation. (iv) The separated bands of DNA are cut and extracted from gel piece. This is known as elution.

Question. Draw a schematic sketch of pBR322 plasmid and label the following in it
• Any two restriction sites.
• Ori and rop
• An antibiotic resistant gene.