Suppose I am performing a restriction digestion on a TRR1 PCR product using the restriction enzyme Hinf1 and I separate the restricted products electrophoretically on agarose gel. How can I expect these results to differ from a virtual or in silico restriction digestion?
You can get star activity (maybe not with this enzyme), You can get only partial digestion so You will see additional bands, but the most important: Your digestion can be influenced by DNA methylation:
New England Biolabs has written that for this enzyme:
Methylation Sensitivity
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Some Combinations of Overlapping
So, if You do not know is Your CG sequence is methylated or not You will newer know if it will be digested or not.
Therefore, in silico is good for preliminary analysis but expect unexpected (once I had everything nicely planned, but got much bigger fragment during analysis than expected as one restriction sites was methylated).
Hi Yasmin, In my study I get same result for agarose and virtual. I use restriction enzymes from NEB and NEB cutter tool (virtual).
Please take note that there will be no methylation on PCR product.
http://tools.neb.com/NEBcutter2/
Are You absolutely sure that it is unmethylated? If yes, than the results should be the same if You do not get partial digestion or star activity doing it in reality.
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