direct sequencing of bisulfite modified DNA - (Nov/10/2005 )
I plan to do direct sequencing of PCR fragment after bisulfite modification and hope to get some quantitative date about the methylation status of individual Cs. What software can be used to quantify C/T ratio in the chromatogram? What I get from our seuqncing facility is an abi file.
Thanks in advance!
if you can get your hot little hands on a SNP program that would certainly help, essentially the CG site is a "SNP" after bisulfite PCR.
I have measured the peak heights manually in the past.
I try direct sequencing of bisulfite modified DNA with the Beckman Coulter Sequencer. My problem is that I don`t know what concentrations of dNTPs and ddNTPs I need to get very good signals. Can anybody help me ?
Does anybody try it with Beckman, too ?
for some strange reason (i think it's nucleotide content) the revers primer is the only primer that would work in a direct sequencing reaction. The forward never works.
Focus your efforts using the reverse primer.
Yes that`s right. The reverse primer works better than the forward primer. For the direct sequencing I used the mix of dNTP/ddNTP from Beckman. But I would make the sequencing by my one, without their kit. And for that I don`t know what concentration of dNTPs and ddNTPs they used. What concentrations do other people use for direct bisulfite sequencing ?
I have never tried that in the past,
however I have directly labelled my PCR amplicons with fluorophore conjugated dUTP and I had to optimise it for the fluorphore,
1:100 and 1:1000 are ratios of flourophore to dNTP that worked well.
In the case of dideoxy sequencing, I am sure if you pull up the original paper, this would tell you the ratios