I am working on a gene which is cloned in a vector which has an internal restriction site for BSU36I. The actual recognition sequence for BSU36I is 5' CCTNAGG 3', and the my gene has 5' GCTCAGG 3'. Is the enzyme specificity going to be affected due to the presence of 'G' instead of 'C' in the 5' side?
HI,
Yes it should be effected. I have been dealing with the same sort of enzyme, even a single base change in recognition site will effect enzyme specificity.
the best is to test experimentally. Atherwise, at first glance it should not work on the mutated site.
Thank you. Yeah I have tried it experimentally and I could not find any linearization of the plasmid. Since, it got only altered by a single base, and the cleavage is going to happen after the second base from the 5' end(ie after second 'C'), I was thinking to increase the enzyme concentration in my reaction mixture and check.
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