Tools Of Recombinant Technogogy

 restriction enzymes, polymerase enzymes, ligases, vectors and the host organism. 

 Restriction Enzymes;

In the year 1963, the two enzyme responsible the growth of bacteriophage in escherichia coli were isolated . one of these to added to methyl group to dna, while the other cut dna the later wa scalled restriction endonuclease.
The first endonuclease-Hind  II, whose functioning depend upon specific nucleotide sequence was isolated and characrectrised five years later it was found that Hind II , today we know more than 900 restriction enzyme that have been isolated from over 230 strains of bacteria.
 
The convention for naming these enzyme is the first letter of the name comes from the genus and second two letter


comes from the species of the prokaryotic cell from which they wewre isolated example EcoRI comes from the Escherichia coli RY 13. In EcoRI , the letter is derived from the name of the strain . Roman number following the names indicate the order in which the enzyme were isolated from that strain of that bacteria . 
restriction enzyme belong to a larger class of enzyme called nuclease. These are of two kind ; exonuclease and exonuclease. exonuclease remove nucleotide frpm the end of the DNA whereas ; endonuclease make cut at specific positions within the DNA.
Each restrictiom nuclease function by inspecting the lenght of a DNA sequence . once itn find a specific sequence , it will bind to the dna and cut each of the two strands of the double helix . 
Restriction enzyme cut the dna a little away from the palindromic sequence sites.

Restriction endonuclease are used in genetic engeneering to form 'recombinant' molecule of DNA , which are composed of DNA from different suorce of genome.
When cut by the same restriction enzyme , the resultant DNAfragments have the same kind of sticky end and these can be joined together end to end using DNA ligases.

Seperation and isolation of Dna fragments ; the cutting dna by restriction endonuclease result in the fragments of dna these fragments can be joined together by a technique known as gel electrophoresis . since dna fragments are negatively charged molecules they can be seperated by forcing them to move towards the anode under an electric field through a medium matrix. nowadays the most commonly used matrix is agarose whch is natural polymer extracted from sea weeds. the DNA fragments seperate resolve according to their size through sieving effect provided by the agarose gel . 

the seperated bands of dna can be visualised only after staining the dna with a compound known as ethidium bromide followed by exposure of UV radiation you cannot see pure dna fragments in the visible light and without staining . you can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV radiation. The seperated bands of DNA are cut out from the agarose gel and extracted from gel piece.This step is known as elution.