restriction enzyme sites - disruption (Aug/11/2008 )

Hi everybody
Can someone tell me how to disrupt
the site of a restriction enzyme.
Thank you for your help

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the easiest and the common way to disrupt the restriction enzyme site is to do site-directed mutation in the recognition sequence. Any changes in the bases within the recognition sequence will work.

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there are three ways to go about this

1- site directed mutagenesis.
This method uses PCR and primers that have a base pair change at the point you are trying to mutate

2- cut the site and ligate it to a short oligonucleotide (linker) that kills the restriction site
For example you have the restriction site

A AGCTT
TTCGA A

this restriction site is ligated to a short oligo nucleotide such as

AGCT CCTTGG

which you can design to add a new restriction site, to help screen for the ligation of the linker

3- you can cut the restriction site and blunt end it with klenow and religate the blunt ends together.

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Thank you all for your help.
I will try it.

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Hello.

Alternatively, you can use dna polymerase with the presence of dNTP to blunt the RE site, and then ligate it. Be sure there is no other blunt end DNA strand in the ligation or else inverted or multiple ligation may happen (can anyone help me to recall the term for "multiple ligation" )

Also, please choose correct dna polymerase due to different exonuclease activity.

(Just noticed this method has already been purposed by perneseblue, hehe...)

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