Identify the tools of r-DNA technology

Explanation

Key tools are:
1) Restriction enzymes : Two enzymes responsible for restricting the growth of Bacteriophage in Escherichia coli were isolated in the year 1963. One of these added methyl groups to DNA and the other cut DNA. The latter was called restriction endonuclease. The first restriction endonuclease - Hind II which cut DNA molecules at a particular point by recognising a specific sequence of six base pairs, called recognition sequence for Hind II. Today, more than 900 restriction enzymes were isolated from over 200 strains of Bacteria, each of which recognises a different recognition sequence.
E CORI is a restriction enzyme in which, the first letter comes from the genus (Escherichia), and the second two letters from the species of the Prokaryotic cell [coli], the letter .R. is derived from the name of strain. Roman numbers indicate the order in which the enzymes were isolated from that strain of Bacteria, Restriction enzymes belong to a larger class of enzymes called nucleases. They are of two types.
a) Exonucleases which remove nucleotides from the ends of the DNA.
b) Endonucleases which make cuts at specific location with in the DNA.
Most restriction enzymes cut the two strands of DNA double helix at different locations. Such a cleavage is know as staggered cut. E CORI recognises 5. GAATT 3. sites on the DNA and cuts it between G and A results in the formation of sticky ends or cohesive end pieces. This stickyness of the ends facilitates the action of enzyme DNA ligase.
Cloning vectors : The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vector. Vectors used for multiplying the foreign DNA sequences are called cloning vectors. Commonly used cloning vectors are plasmids, bacteriophages, cosmids. Plasmids are extrachromosomal circular DNA molecules present in almost all bacterial species. They are inheritable and carry few genes are easy to isolate and reintroduce into the bacterium (Host).
Features required to facilitate cloning into a vector :
a) 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 host cells. I is also responsible for controlling the copy number of the linked DŅA.
b) Selectable marker : In addition to .ori., the vector requires a selectable marker which helps in identifying and eliminating non-transformants and selectively permitting the growth of the any transformants normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin etc., are useful selectable markers for E.Coli.
c) Cloning sites : In order to link the alien DNA, the vector needs to have very few, preferably single recognition sites for the restriction enzymes.
d) Molecular weight : The cloning vector should have low molecular weight. .
e) Vectors for cloning genes in plants and animals : The tumour inducing (Tl) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector such that it is no more pathogenic to plants. Similarly retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.