Nested PCR for BSP question - (Jun/01/2007 )
Hi Quick question,
This may seem silly, but would there be any inherent problem with a first round nested PCR amplification of 40 cycles and a second round amplification of the BSP product of 25 cycles? This would be using fully nested primers.
Depending on what you start with you will probably want to dilute after the first round to add to the second round so there is not too much template.
I normally don't have to dilute as I do the nested strategy to cope with the low concentration of DNA after Bisulfite. I use 1 µl of the reaction mix of the first round in a 25µl reaction for the second round.
cancergeek, there could be inherant problems in using so many cycles.
The major one would be PCRbias, if in the intial round a certain subpopulation is preferentially amplified over another (as a function of amplicon base composition) then these molecules would be over-represented in the second round of PCR (for example you see more methyluated amplicons (higher CG content) than unmethylated (high AT content) after sequencing).
We get around this by performing replicate PCR reactions from the one sample and then pooling them fo subsequent cloning and sequencing.
I have used 1-3 ul of first round PCR products as template in the second round PCR, and they thow no significant difference.