Test: Tools of Recombinant DNA Technology (January 19) - NEET MCQ

When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of 'sticky-ends' produced, which can be joined together (end-to-end) using DNA ligase. Restriction enzymes are of two kinds - exonucleases and endonucleases. Exonucleases remove nucleotides from the j ends of the DNA whereas endonucleases make cuts at specific positions within the DNA. Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning. Therefore, in order to link the alien DNA (or foreign DNA), the vector needs to have very few, preferably single, recognition/cloning sites for the commonly used restriction enzymes.

HindII was the first discovered restriction endonuclease. It was isolated from Haemophilus influenzae. It produces blunt ends.

• Electrophoresis is a technique used for the separation of molecules based on their size and charge.
• Plasmids are extra-chromosomal, self-replicating, usually circular, double-stranded DNA molecules found naturally in many bacteria and also in some yeast.
• It is not advisable to use an exonuclease enzyme while producing a recombinant DNA molecule.
• In EcoRI, the roman numeral I indicates that it was the first enzyme isolated from E.coli RY 13.

A tumour inducing Ti plasmid of Agrobacterium tumefaciens has been modified into a cloning vector which is not pathogenic to the plants, However, it is still able to use its mechanism to deliver genes of our interest into various plants.

The cloning vector requires the presence of a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc. are considered useful selectable markers for E. coli. The normal E.coli cells do not carry resistance against any of these antibiotics.

Vectors that have high number per cell will have high copy number of their genome within the bacterial cell. If we link an alien piece of DNA with vectorm we can multiply its number equal to the copy number of the vector. Any piece of DNA when linked to the 'ori' sequence, can be made to replicate within the host cells. This property of 'ori' is used to make a number of copies of the linked DNA. If we want to obtain many copies of the target DNA, then it should be cloned in such a vector whose 'ori' supports high copy number.

Transformation is a process by which a cell takes up naked DNA fragment from the environment, incorporates it into its own chromosomal DNA and finally expresses the trait controlled by the incoming DNA. Since DNA is a hydrophilic molecule, it can not pass through membranes, so the bacterial cells must be made competent to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium (Ca²+) which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.

Alternative selectable markers have been developed which differentiate recombinants from the non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinantDNA is inserted within the coding sequence of an enzyme, β-galactosidase. This results into inactivation of the enzyme, whcih is referred to as insertional inactivation. The presence of a chromogenic substrate given blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

Micro-injection method is the direct or vectorless method of gene transfer, in which foreign DNA is directly injected into the nucleus of animal cell or plant cell by using micro-needles or micro-pipettes. It is used to transfer DNA in oocytes, eggs and embryo.

Biolistic method or gene gun method is a direct or vectorless method 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 at a very high velocity.