Bisulfte PCR and sequencing PCR and Primer Notes - Tips and hints on PCR for bisulfite converted DNA and bisulfite primer (Jan/08/2007 )
Hi! I am new in the field and I am trying to design primers for bilsulfite pyrosequencing. I tried the Meth Primer but the problem is that the primers designed by the programme are not of my interest. I understand that when there are many CpGs, the programme avoids that region because the primer should not contain many CpGs. But If this is the region of my interest, is there any possible way to overcome this problem?
And another question:
How can I design the primer for pyrosequencing?
Thanks for your informations about bisulfite PCR.
I am a student majoy in methylation of DNA. now I 'm quite confused by my experiment. i'm doing bisulfite sequencing and now the PCR products is not what i want.
the sequence in the agrose gel (2%)is not the target seqence, my target sequence is 243bp, but in the gel, it's may be 270bp.(fig1)
but more confused me is that i use 8% PAGE gel run my product, it indicated about 330bp(fig2). i don't know why?
can you help me?
This is going to be a little long I am designing primers for both bisulfite sequencing and MSP. I have a few questions:
1) For bisulfite sequencing, I will amplify the region spanning the CpG island. I am designing primers in regions where there is no CG pairs. At the same time, I am trying to keep the Tm around 60C, which means I have to use longer primers (30-40 bps). Do you think this is a good idea? Since there are only three bases in these primers, I think primer dimers might be a problem, but I don't want to use shorter primers, which will have low CG content, thus lower Tms. Any recommendations?
2) I am designing primers for MSP. I try to include at least 3 CGs in each primer, with at least one pair at the 3' end. Should I choose the same CG pairs in both sets of primers? I mean, should primers that amplify the unmethylated DNA and the primers that amplify the methylated DNA inlude exactly the same CG pairs? I've seen in the literature that they use primers on different regions of the CpG island. But if we are looking at the methylation status of the promoter, we should basically look at the same CG pairs in order to prevent bias. Is this correct?
3) I've already done this analysis on one promoter with primers that we obtained from our collaborators. I just repeated what they did on a different cancer cell line. In the MSP, I see bands in both PCRs. How could this be possible? Does this mean I have a mixture of methylated and unmethylated DNA in the genomic DNA extract, or could this be because of the poor primer design or maybe incomplete bisulfite conversion? What do you do when you see fragments of expected size in both PCRs of MSP?
Thank you for any ideas in advance.
1) Absolutely, there is not need to really worry about primer dimer etc because you are designing strand specific PCRs
2) yes ideally you want to design your primers for MSP this way, this may mean you may need to add one or two bases at the 5' end.
3) you may have saturated the system, or the Tm is too low so both primers are binding "non-specifically"