I am running inverse PCR to try to sequence the region flanking a novel gene. My question is whether or not it is OK to use a restriction enzyme that cleaves my known sequence at one site. That is, does my template need to be circular? It seems like it wouldn't matter since the the primers are pointed out away from the cleavage site.
Let me clarify my question. My understanding of inverse PCR is that you digest genomic DNA (pre-PCR) with an RE, then ligate with ligase, followed by PCR using inverse primers pointing away from the known sequence. The cleavage and ligation will have created random circular DNAs and iPCR will amplify the two regions flanking the known sequence that have been joined by ligase. But I read somewhere that I should avoid an RE that cuts in the known sequence, making it not circular but linear. I don't see why that would be a problem since the amplified region would be the same. So, is it OK if the enzyme chosen cuts once in the known sequence?
Okay, as far as I am understanding before PCR the RE used for digesting the genomic DNA should cut ONLY in the flanks and not the known sequence. Otherwise the ligation would result in a MIXTURE of randomly ligated linear fragments. In the correctly oriented linear fragments both the primers would point away from the known sequence but towards each other and result in the amplification of the ligated flanks. In the incorrectly oriented linear fragments there are multiple types of ligated products where the orientation of primers may be in the same direction for both flanks or opposite to each other. Concatemerization would also occur much more frequently in linear fragments compared to circularisation.
So, in my humble opinion the RE used for gDNA digestion before PCR should NOT cut the known sequence.
Dear David,
avoid to use a restriction enzyme that cuts the known sequence. Your assay will not work in case your primers are on opposite sites since you will generate circles with only one primer target site. In case primer sites are not separated by the site you will simply loose information.
Best, Uwe
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