PCR bias with BSP primers - (Jul/02/2008 )
Hi,
What I want to be able to do is design primers for RT-PCR and HRM analysis to look at methylation of CpG islands. I would also then like to sequence over that region to check the methylation of my methylation controls and any samples that look interesting. To avoid PCR bias I was thinking of designing BSP primers but from this study ( Biotechniques. 2006 Sep;41(3):274, 276, 278. Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay.Wojdacz TK, Hansen LL.) it appears as though this still preferentially amplifies unmeth template. Is this correct? i don't understand this nor do i understand how adding a C in 5' end of each primer helps to amplify a methylated template if you don't know the methylation status of that site in the meth template. Can someone please explain.
Cheers.
What I want to be able to do is design primers for RT-PCR and HRM analysis to look at methylation of CpG islands. I would also then like to sequence over that region to check the methylation of my methylation controls and any samples that look interesting. To avoid PCR bias I was thinking of designing BSP primers but from this study ( Biotechniques. 2006 Sep;41(3):274, 276, 278. Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay.Wojdacz TK, Hansen LL.) it appears as though this still preferentially amplifies unmeth template. Is this correct? i don't understand this nor do i understand how adding a C in 5' end of each primer helps to amplify a methylated template if you don't know the methylation status of that site in the meth template. Can someone please explain.
Cheers.
Someone may give you specific answer soon, so keep checking! In the meanwhile, see these methylation related protocols for any hints:
http://search.vadlo.com/b/q?sn=158621799&a...ation&rel=0
hth/