Sequencing bisulphite modified PCR- Very bad sequence?? - (Nov/24/2004 )
we have been trying to opitimise this bisulphite method for sequencing promoter of our gene of interest. We are using the kit from Chemicon and did get it to work successfully for some control samples. Which when PCR'd looked nice and clean. Subsequently for patient material (tumour tissue extracted from paraffin) it has not worked i.e no bands in the PCR.
However I did sequence the controls which were successfully amplified and the sequence looked terrible. Very bad bacground, overlapping sequence, basically unreadable.
Does anybody have any suggestions for why the sequencing looks so bad?
Also, has anybody been doing this with DNA extracted from paraffin and do you have to modify the protocol??
Any advice would be appreciated
If you have got nice PCR bands you should be able to get nice sequencing results too. Here are some things you may want to check for:
1) Have you got sufficient amount of DNA? Although quantitation of PCR products is not necessary, make sure the bands are strong and clear. If not, use bigger volume such as 50 ul for PCR reaction or combine two reactions into one.
2) Have you purified your PCR products? If PCR bands are clear and there is no non-specific bands, purify the PCR reaction using Qiagen PCR purification kit, if not, run a gel to separate the bands and recover the DNA using Qiagen gel purification kit. We have also used ExoSAP IT from USB to purify PCR products before sequencing, which involves a 15 min digestion and is much simpler than other methods, and the results are generally good.
3) Use downstream primer for sequencing, which gives better results than upstream primer.
4) Follow requirements set by your sequencing facility for preparing sequencing reaction.
5) For DNA from paraffin section, keep PCR product size as small as possible (<200 bp).