Primers for BS treated DNA - (Nov/17/2006 )
I've read and had discussions with a colleague, and got to know that most of the time, primers designed for Bisulfite treated DNAs are for rather short fragments, 200-300bps or maybe up to 400-500bps. Longer stretches pose a problem as they might break easily.
Could anyone give me some advice on how to avoid this? We've design a primer for a stretch of about 1kb (not that extremely long i think), the problem we face is that the product sometimes appears and sometimes not. We suspect that the BS DNA may have been degraded partially, as there's a product when used as a tempate with a primer pair for a 400bp product.
bisulfite degrades DNA so that is why primer design for smaller fragments is required.
I have amplified a 1.2kb fragment in the past, but that was as long as I would go.
What PCR amplification cycle are you performing? A nested strategy with two rounds of PCR would certainly help in yielding your amplicon of interest.
We've actually tried both nested and touchdown.
We weren't successful with nested at all.
Some luck in touchdown, but it's all the time inconsistant -- between different batches of BS DNA as well as between colleagues.
The annealing goes from 56oC- 50oC (1 cycle each) and then 49oC thereafter for 33cycles. We couldnt get a better set of primers with higher Tm unfortunately
just out of interst sharonpek,
how were your primers designed? were they by eye or with a program?
Hmm, although it has not been updated recently, perlprimer does pick primers that are optimal for bisulfite PCR.
You could try lowering your Tm in your PCR to up to 6C below the calculated Tm (each time in 2C increments) and see if you get consistency that way.
Another parameter that you can play around with is the magnesium concentration in your PCR, a titration of magnesium (0mM-3mM, in 0.5mM increments) with set annealing temperatures would also help as sometimes the generic 1.5mM MgCl2 may not be efficient in all cases. If these are assays you intend to run routinely then I think it's worth the effort in optimising the PCR.
a 1kb fragment is on the high side but amplifiable. I tend to try and pick primers that generate a final amplicon of 600-800bp as this is the limit of a good sequencing run.