high GC content in PCR? - (Jan/18/2006 )
Does anyone know the explanation behind the trouble doing PCR on samples with high GC content? I have found plenty of solutions to help fix the problem, but not a real answer to why it is a problem in the first place. Thanks.
I think it may have to do with the higher melting tempertature of high GC content sequences that makes it more likely for mispriming to occur...
also possible problems with secondary structure??
Don't know for sure, maybe someone else knows more of the chemistry??
Secondary structures formed by high GC DNA is harder to melt due to the extra hydrogen bond that G-C pairs contain (A-T pairs only contain 2). As a result, higher temperature or reagents such as betaine or DMSO are required to help break up these secondary structures. This allows the PCR to go ahead as normal. Also, with high GC genomic DNA, it is advisable to have a hot start with a long initial melting step of 10 minutes to ensure that the DNA is fully melted.