systemic way to reduce BSP PCR bias - (Mar/13/2007 )
we found a significant PCR bias dependent on annealling temp, so in our lab, we dod systemic way to optimize PCR condition for Bisulfite PCR followed by pyrosequence. i would like share my experiance.
theoretically, bisulfite products is mixture with C/T SNP templete, the template contain more C will have better binding ability (the affinity between C-G is higher than T-A), and they require a higher Tm. that is the reaosn we see a significant PCR bias dependent on annealling temp.
then we make standard control by genomiphi and SSSI, after 3 round genomiphi genomic amplification, the DNA contain 0 methylation; after SSSI, the DNA contain 100% methyaltion, then we mix them by equal ratio to produce a 50% methyaltion, then do bisulfite treatment.
we check 30 primers using this control, we finally figure out we should optimize PCR conditon all by this way. you wil see that clearly by this two figure. generally we recommand to use lower temp if primer can work well.
Thanks for sharing, Gaodaxia. There are alos some reports on using primers containing one 5' CpG C to avoid bias. Any experience with that?