Direct sequencing on BSP PCR products: problems with A and T stretches - (Nov/08/2006 )
I just started direct sequencing on BSP PCR products and I got mixed results. The PCR were working well and I used the same primers for sequencing, and most of the time, I got both forward and reverse primer to work, at least I cannot say, like some people did on that forum, that the forward primer is working better than the reverse. The problem I am facing is when the Taq polymerase has to go through a stretch of T or A. When there is at least 10 A or T, then afterward, I got double peaks, probably because of slippage of the polymerase. I am using ABI Big Dye Terminator kit and an ABI 3730 sequencer. In some cases, I think I can design other primers to circumvent this problem but it is not always possible. And this will dramatically increase the number of sequencing reactions I have to do.
Does anyone know how to solve this problem ?
In my experience this usually happens not in the sequencing reaction, but in the original PCR reaction. Try lowering the extension temperature during the BSP PCR reaction -- I would be using 64C maximum, with a little longer extension time. The problem can actually also be that the organism has variability in this region, since the in vivo polymerases also have trouble in this case.
I guess you were trying to say the revers primer works better than the forward, which is true.
I don't think long stretches of As or Ts cause any problem. Sometimes if a sample lacks methylation, unused C or G dye may appear as background peaks. This is an inherent problem. To overcome it, some people tried to introduce some extra Cs (forward) or Gs (reverse) into the 5'-end of BSP primer to balance the scantness of C or G. Other problem can be eliminated by optimization of sequencing reaction such as fine tuning template and primer amount, etc.
Well, in my case, stretches of A and T are a real problem ; besides that, I got very nice sequencing reactions. I tried lowering the extension temperature to 64°C, it is slightly better in one case but it is not a big change. Maybe I will try to lower the number of cycles since I have a good PCR efficiency. I dont think it can be a problem of variability in the organism since these A/T stretches are a consequence of the bisulfite conversion. But actually, I am not so surprised because the same thing happens on "traditionnal" sequences when the polymerase has to go through a repetitive sequence, like a microsatellite for example. I am just surprised to hear that I am the only one facing this problem.....