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restriction enzyme query - (Mar/23/2010 )

restriction enzyme does not harm bacterial DNA or we can say that it cannot cut the bacterial DNA
then hw can it <restriction enzyme > be used to cut gene in plasmid in recombinant dna technology bc plasmid has taken from bacteria and restriction enzyme doesnt harm bacteria

-aaa-

aaa on Mar 23 2010, 10:29 AM said:

restriction enzyme does not harm bacterial DNA or we can say that it cannot cut the bacterial DNA
then hw can it <restriction enzyme > be used to cut gene in plasmid in recombinant dna technology bc plasmid has taken from bacteria and restriction enzyme doesnt harm bacteria



Could you explain more on your question? I dont understand your points.

-newborn-

Each restriction enzyme has a methylase associated with it on the chromosome. This methylase puts methyl groups on the host DNA, and the restriction enzyme doesn't recognize its recognition sequence when it is so methlyated. The host DNA is thus protected from the actions of its own restriction enzyme.

Incoming (foreign) DNA is unlikely to be protected (methylated) in the same manner, thus this invading DNA is digested by the hosts restriction enzyme(s).

When working in cloning experiments, the principle is the same -- DNA to be digested is carried by a plasmid in a host that does not methylate DNA in the pattern that would cause the restriction enzyme to see it as protected, thus it is cut. DNA generated by PCR is similarly unmethylated, and is therefore also digested.

Some enzymes won't cut DNA isolated from dam+ or dcm+ hosts (two common bacterial methylases), thus one must know the genotype of the host cloning strain if using a restriction enzyme whose action is blocked by dam or dcm methylation.

-HomeBrew-

thanks a lot !

-aaa-