amplifying small amounts of converted DNA or unconverted DNA? - (Jul/09/2008 )
I'm working on the PCR of some samples I have bisulfite treated using quiagens EpiTect bisulfite kit. From the first PCR I got no visible bands after running a gel. Then I used that PCR as a template and was able to see a band from 3 samples (out of 8). I did another PCR using the second PCR as a template to further amplify anything that may be there. When I ran a gel out for those PCRs I got a band for every sample, some more intense than others. My question: do you think my bands are of converted or unconverted DNA? Is the only way to determine which it is to sequence? Each PCR had 35 cycles with an annealing temp of 48C. The primers are not converted DNA specific (they only contain a few C's that would be converted and since the annealing temp is so low my PI insists that I do not need to have conversion specific primers- quiagen also insists their DNA conversion rates are greater than 99.5%). Do you think I just got a super low yield after bisulfite treatment and it took a couple rounds of PCR to amplify my target or that i have amplified that 0.5% of DNA that may have not been converted?
Also, I have an additional target I would like to PCR out of bisulfite treated DNA but it is quite large, around 1.5 kb. Am I over reaching with this large of a product? What is the max size product I would realistically be able to PCR out of bisulfite treated DNA?
Thanks for taking the time to read, any help would be appreciated!
First of all I think it is odd that it took three rounds of PCR to amplify the product. If there was enough template from the 2nd PCR (in what, 2ul?) that it amplified in the third round, it really should have amplified in the 2nd round. Remember that a PCR, if 100% efficient will cause a doubling in the amount of product each cycle. Perhaps you need to play around with the PCR conditions?
Re: converted or unconverted, that would depend on your primers. If they contain lots of non-CpG C's, then it would suggest it is converted template. I'm sorry I didn't totally understand what you meant when you said, "The primers are not converted DNA specific (they only contain a few C's that would be converted.." as this seems contradicting. But if they contain only non-CpG C's, you should be getting the converted strands.
1.5kb is very large, but probably not beyond the realms of possibility since the Qiagen kit claims to not cause degradation of the DNA. Why not just break it up into a more reasonable 2 or 3 fragments?
Hope this helps you!
one way to determine if your amplicons are of converted or unconverted template is to do a restriction digest to test for the loss of the restriction site,
one I choose is that of MspI which recognises CCGG, so my question would be, does your region of interest have one or more of these sites? if your amplicon is converted then you will lose the site and see no digestion, if you get digestion then you have amplified unconverted template.
I suspect with the low Tm, the three cycles of PCR in combination with your primers, you have unconverted template.
one other question, with the successive rounds of PCR, were you using a nested PCR strategy?