A problem with bisulfite sequencing - (Aug/19/2006 )
Hello, everyone. Recently I've been doing the bisulfite specific PCR and sequencing. Everything went well untill I came upon the step of sequencing of positive clones. The inserted fragment incompassed too much A&T(>70%) due to the C to T conversion so that the sequencing company only got 1/3 of the clones fully sequenced. It said that this kind of PolyT structure was a major obstacle for the fluorescent-labeled sequencing method. I chose several companies and the results turned out to be the same. I wondered whether anyone here had come across problems like mein. By the way, I am in China. Maybe the companies are not so high qualified. I shall appreciate that if someone would like to give me some advice.
hi pan lei,
what sequencing vector are you using?
I use pGEM from promega and have sequenced using T7, SP6 and M13 primers, I found that SP6 worked better in that longer runs were achieved.
I used ABI Big Dye version 3.1 (1/8th strength) and they were run on a capillary sequencer.
Could you post up a chromatogram for all to have a look at?
hi,Nick. Thanks for your attention.
I also use pGEM-T, but I have only sequenced using T7 primer.
Does SP6 work better disregard of the composition of the sequence inserted or because the sequencer handle better with PolyA than PolyT? Because I found it peculiar that most of the sequences I got by sequencing with T7 primers was the same as my pcr template but not the complementry strand. But aren't the chances of forword and reverse ligation equal, at least theoretically?
I will try with SP6 primers. I am not sure which dye( or software?) was used. But one company used the ABI 3730XL sequencer.
Here I post up one chromatogram of unsuccessful sequencing(the forum said I can't post a file with .abi extension, so I just post part of the graph as a picture), hope that would suggest something.
Based on my experience sequencing hundreds if not thousands of cloned bisulfite PCR products, I think once your get your PCR product into a vector, sequencing is not a problem although direct PCR sequencing is very challenging. Sometimes a few samples do come back with bad result just as what your example shows, I found the failure is almost always caused by bad plasmid purification.
I don't know if different vector systems differ in terms of giving good sequencing results, but I have been always using invitrogen TOPO TA cloning vector. I have used at least three sequencing facilities, all of them give comparable and satisfactory results even without special handling of my samples which are prepared according to their requirement for sequencing plasmid. I give them the right amount of plasmid and specify the standard primer to be used. They will get the job done.
Some companies provide full service sequencing which means you just give them your purified plasmid, they will check whether your DNA is pure enough, what amount to use in sequencing reaction etc. If you have access to such service, give it a try.
If you are in the US, try MCLAB at www.mclab.com. They do a very good job sequencing uncloned bisulfite PCR products, let alone those in a plasmid.
This seems to be similar to the problem I was having a few weeks ago. I found that T7 didn't work very well as a primer. I tried M13F and M13R (using TOPO vector) and found that sequencing the strand that contained all the A and C nucleotides worked much better for my particular sequence. I've actually started using the reverse primer used for PCR as the sequencing (no CpGs right next to primer site so I don't lose any information).
Hi,everyone. Today I got my sequencing results back. All my 8 clones seqenced with SP6 primer gave quite clear and fine chromatograms. And I am looking forward to receive quite consensus data after sending all the positive clones I've checked. Thanks you guys, methylnick, pcrman and ultragirl.
I also have troubles with sequencing. In fact, I'm using a Quiagen kit for bisulfite treatment, and after I carry out a nested PCR, primers was taken from a paper, I checked them on the NCBI BLAST. running the second PCR on a gel, I get poor bands but all in the same size as I expected. Then I cloned them into a pCR2.1-TOPO vector, using the TA TOPO cloning kit. After that we send it to sequence but we got back poor results. I do the plasmid purificaton with the Quiagen miniprep kit so I don't think it won't be insufficient. Do you have any suggestions what should I change?
could you elaborate on what you mean about poor bands and poor sequencing results?
could you post scans of you PCR gel and a chromatogram for us all to see what is going on?