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Bisulfite sequencing and PCR issues


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#1 spjgjsm

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Posted 17 September 2004 - 01:02 AM

Hi

I'm trying to do some direct sequencing on a number of bisulfite-treated DNA samples from different tissues (blood, brain, buccal). I've been using the CpGENOME kit for the bisulfite treatment starting with 1ug DNA. This seems to be working fine as my bisulfite-specific PCRs are working fine on the resulting DNA from all tissue types. My average PCR product sizes are 300-400bp and there appears to be no non-specific amplification. So far, so good.... Sequencing the products is another matter, however. The blood DNA is sequencing great and giving me good data. The DNA from the other two tissue types is proving a lot harder....Can anyone give me any help / advice? Could it be that the DNA from the blood / buccal is not clean enough? Should I alter the cycling conditions, or increase the amount of starting template..?

A related question - given that the bisulfite-specific PCRs are working on all samples (i.e. my the bisulfite-treatment is fine and the PCR assay is fine), am I correct in thinking that even though I can't get these to sequence well, they should still be ok for a SNuPe assay??

Any help would be gratefully recieved!

Best wishes

Jon

#2 pcrman

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Posted 17 September 2004 - 05:20 PM

The blood DNA is sequencing great and giving me good data. The DNA from the other two tissue types is proving a lot harder


Do you mean you cannot get good (readable) sequencing results? How do the PCR bands look like for those samples? Are they the same as bands from blood samples? Certainly, your starting materials will influence your PCR results which in turn influence sequencing results.

My feeling is that MSP is easier than getting good templates for sequencing. So not getting good sequencing results dosen't necessarily mean that your modified DNA is not good.

Edited by pcrman, 18 September 2004 - 10:51 AM.





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