nested PCR after bisulphite - (Aug/24/2005 )

hello all!

congratulations for the website, it is an excellent tool for scientists
I was wondering why do you all use nested PCR for bisulphite treated DNA? because i think there is an increased risk of PCR bias using 2 rounds instead of one round, what do you think? I've changed for 4 hours incubation in order to get amplification on the first round.
another question: how long would you store treated DNA in an agarose bead? because I had trouble amplifying after 2 weeks of storage at 4ºC

Thanks!

Cris

Hello cris,

the nature of bisulfite PCR is that you are already biasing the amplification with the primers that you select. They are strand specific and you want to bias towards fully converted DNA molecules.

The main reason for two rounds is that the starting material is so low that you require two rounds of PCR to obtain a workable amplicon.

I have not had much experience with DNA in agarose beads, its such a tedious method!!

Nick

I agree with Nick.

For both BSP and MSP, nested PCR is preferred if not required. As Nick said, the reason is the starting material is extremely low. For BPS, if you don't have nested primers, you can just carry out two rounds of amplification using the same set of primers, or one round PCR with additional Taq added after 40 cycles.

For MSP, the first round PCR is carried out using universial primers (or BSP primers, no CpG Cs in primer) and the 2nd PCR using MSP primers. It is not a good idea to run two rounds of PCR using MSP primers only.

Hello!

Thank you for your replies; I agree with you both but I still think that it is better if you can amplify with only one round (for BSP). My experience is that, in one of the genes that i can distinguish the two alleles, it is most probable to loose one of the alleles with two rounds instead of one round PCR. But this could be due also to the small amount of DNA that i'm using currently (200-300 cells!), actually that's why I'm using the tedious agarose bead protocol (and that's why it gets degraded so fast at 4ºC, I think!)

Cris