3 replies to this topic

[Lj Lee]

Hi all,

 

And idea on how to analyse data for direct sequencing of PCR products? Are there any software or online tools available for us to do so?

 

Many thanks  

[phage434]

You should be able to sequence with one (or the other) of the pcr primers. You won't see the first section of sequence, but if it is short  enough, you can see both ends by sequencing twice, once with each primer.

[Lj Lee]

You should be able to sequence with one (or the other) of the pcr primers. You won't see the first section of sequence, but if it is short  enough, you can see both ends by sequencing twice, once with each primer.

Hi phage434, 

 

Thanks for your reply. Yes I got the sequence for both forward and reverse sequences and I aligned these two together by reverse complimented the reverse sequence. My question here is, because I direct sequenced the PCR product which is heterogenous, I had multiple sequences in some position, I heard that there are some ways that we measure the peak of the eletrogram to measure the percentage of methylation at that particular position, any idea how to do it?

 

Thank you very much.  

[phage434]

I don't know of a quantitative method for determining this, other than cloning the fragment, sequencing several clones (which should not exhibit variation) and doing statistics. If it is a single ambiguous  point, you might be able to set up a qpcr or digital PCR reaction which selectively amplified one or the other sequences. I think the best you can do with a single Sanger read is to measure the two peak heights and estimate a ratio, but many many things could make this highly inaccurate.