3 replies to this topic

[jimmyhj]

Hey,

I am analyzing the methylation status of two regions: one ecompasses 500bp, the other is around 800bp. Could I just designed primers at flanking sites and amply these two regions? I found usually only 200-300bp were used to amplification for Bisulfite sequencing in most referecne. Is there any PCR length limitation for this method.

Thanks a lot ,

Jimmy

[rassen]

I have not tried PCR size bigger than 450 bp but have seen in one paper that over 1 kb was amplified from bisulfite modified DNA. I doubt about it.

[labtechie]

I follow the advice of most authors that 300 bp is about as large as one can go because bisulfite modified DNA is very fragile.  You may want to do a nested PCR to increase product-- your first product might be 500 or 800 bp but you might not see a visible band.  Also it's ok to just know the status of 300 bp or so if you are seequencing a CpG island-- it will give you a taste for what you are looking at and you can amplify the rest of the region separately if you think you have something significant.

[cryobeer]

jimmyhj, on Nov 29 2004, 01:16 PM, said:

Hey,

I am analyzing the methylation status of two regions: one ecompasses 500bp, the other is around 800bp. Could I just designed primers at flanking sites and amply these two regions? I found usually only 200-300bp were used to amplification for Bisulfite sequencing in most referecne. Is there any PCR length limitation for this method.

Thanks a lot ,

Jimmy

We have successfully amplified fragments biger than 700 bp for bifulfite modification based PCR.

Use high quality kits to extract DNA and the fragile problem following bisulfite modification shoudl be decreased and then it should be possible to amplified lagger fragments compared with published papers.

Good luck.