Restriction Endonucleases...?

Question

what chance of a restriction enzyme (eg. Bgl2) cuts a DNA site lacks one bp (eg. AGATCC instead of original site AGATCT).

I also know the theorotical facts, their specifity etc. ; but i need practical facts... i need answer from someone uses restriction enzymes in lab... like a graduate student in molecular biology... but thanks anyway :)

Answer 1

Hello,

It's true what the others have said here, that the enzyme's specificity will prevent most non-specific cutting. However, it's definitely possible for the enzyme to cut in an area that has a one base substitution. In the example you gave, a T is replaced by C and because these are both pyrimidines, the enzyme may still cut due to structural similarities.

If you're having trouble with this type of non-specific cutting, you can try increasing the digest temperature (I guess 37C is Bgl2's optimum temperature...if so, try 42C but nothing higher than that) and decreasing the digest time. Most people do overnight digests, but I haven't had the best luck with that because I get DNA degradation and some non-specific cutting. Try digesting for 2-3 hours and maybe that will make a difference.

I hope this helps some!

Answer 2

Low to zero chance if you work in the optimum time and condition. Restriction enzymes are rather specific enzymes. They should only cut sites that are they are supposed to. If they do not find any, usually they would not cut. However, if you leave the DNA at the enzyme condition for too long, they may start cutting sites that looks like the one that are supposed to. This is where restriction endonuclease starts to cut something like what you mention.

Answer 3

From what I understand, the chance of this happening is extremely low. Restriction enzymes are incredibly specific.