Different methylation with normal Taq and High fidelityTaq - Should I use non-fidelTaq instead of High fidelity (May/03/2005 )
Hi , I met with some problems when I sequenced my BSP PCR product, can anyone here help me out? Thank you very much in advance!
When I used Expanded high fidelity Taq(from Roche) to amplify the Bisulfite-treated DNA, the ampification efficiency is much high than that by non-high fidelity taq, but when I sequence the same fragement amplified with high fidelity taq and non-high fidelity taq respectively, I found the great difference in the methylation status between high fidelity taq and non-high fidelity taq, is the difference due to the proof-reading activity of high fidelity taq which leads to the reversion of T to C? Is that necessary that we should use non-fidelity taq instead of high fidelity taq to amplify the bisulfite-treated DNA?
Looking forward to hearing from some experts here ASAP.
That is a very interesting observation. Could you tell us what differences you have got, do the differences only occur at CpG sites, which one got higher methylation?
I don't think that the high fidelity taq converts T (actually U) back to C. The problem is unmethylated Cs are converted to U and the modified DNA is actually similar to RNA. Probably the high fidelity taqs have problem reading the U (they are intended for DNA). Just a wild guess, I'm not sure. I would suggest you use JumpStart Taq from sigma which doesn't have this problem with giving you very high amplifying efficiency.
that is very interesting and I would say, publishable in as a techniques paper!
Be interested to see what the differences were. I would say that because BSP isnvolves amplification of a heterogeneous population of DNA molecules with varying sequences (with resepct to conversion) I would say that using a HiFi Taq would not give an accurate result and mask the conversion events.
Hi PCRMAN and Nick,
Thank you very much for your prompt reply.
I found that the methylation in my DNA sequence is almost totally different between high fidelity taq-amplified and conventional taq-amplified PCR fragment. They are messed up. I even found there was a big difference between different clones in the same PCR product amplified with high fidelity taq.Besides the high fidelity taq, do you think it may also be due to the incomplete bisulfite treatment or over-treament with bisulfite? How can I know that my DNA is properply and completely treated with bisulfite?
I will use sigma's jump hot start taq.
Thank you very much.
I am still not quite understand what your situation. What do you mean by saying "They are messed up"? You mentioned "different clones", have you done cloning before sequencing? I guess what you really want to say is that the sequencing result are not good, not conclusive..., yes, that's a common problem with direct sequencing. You can do two things if you have not done so: sequence using the reverse primer and do a TA cloning before sequencing.
If you couldn't find a consistent difference in HiFi taq amplified sequence, the problem may not be related to HiFi.
How to check if DNA is fully converted? A simple way is to check if there are non-cpg 'C's not being converted to 'T'. Usually, if the conversion is incomplete, you would see methylation ('C') at non-cpg sites.
Yes, over-treatment could be another problem which will convert 5mC to T.