from which strand to derive BSP primers? - (Apr/04/2006 )
sorry for the basic question. did not find the topic on the archive.
say one has derived a sense primer sequence straight from a modified 'forward' DNA strand.
at PCR cycle 1 this sense primer should anneal at its place on the modified reverse strand, and should extend creating the distal antisense primer binding site somewhere downstream.
I don't dumbly get why one uses antisense primer sequences (MethPrimer, PerlPrimer) complementary to the modified 'forward' strand, rather than complementary to the extension of the sense primer.
The extension of the sense primer is not equal to the modified forward strand (but complementary to the modified reverse strand) since bisulfite treated DNA strands are not complementary anylonger.
It is like if these primers (MethPrimer, PerlPrimer) would extend on original [modified] strands only, and one should think on some kind of PCR recombination to get any primer extending on recent opposite primer extensions.
I know the answer should be obvious but still don't see it.
If I were not completely wrong but still there were advantages doing it the MethPrimer/PerlPrimer way, could anyone comment why?
thanks for your kind clarification.
javier
say one has derived a sense primer sequence straight from a modified 'forward' DNA strand.
at PCR cycle 1 this sense primer should anneal at its place on the modified reverse strand, and should extend creating the distal antisense primer binding site somewhere downstream.
I don't dumbly get why one uses antisense primer sequences (MethPrimer, PerlPrimer) complementary to the modified 'forward' strand, rather than complementary to the extension of the sense primer.
The extension of the sense primer is not equal to the modified forward strand (but complementary to the modified reverse strand) since bisulfite treated DNA strands are not complementary anylonger.
It is like if these primers (MethPrimer, PerlPrimer) would extend on original [modified] strands only, and one should think on some kind of PCR recombination to get any primer extending on recent opposite primer extensions.
I know the answer should be obvious but still don't see it.
If I were not completely wrong but still there were advantages doing it the MethPrimer/PerlPrimer way, could anyone comment why?
thanks for your kind clarification.
javier
Sorry, I am not really replying to your request but in fact, I am completing it since I am also trying to figure out how to design primers for PCR on bisulfite treated DNA. Actually, my problem is first to understand how can a sense primer designed from the modified forward strand can anneal since it is not complementary to the reverse complementary strand (as far as I understand, because after bisulfite treatment, both strands are not complementary any more, am I right ??). I agree with you about the fact that the antisense primer should logically be complementary to the extension of the sense primer and not to the modified forward strand.
Can anyone clarify that for us, maybe with an example ?
Thanks in advance.
Valerie
Valerie
I think I saw why it is the way it is currently done.
It is like if the sense primer does not have much work to do on the first PCR cycles, since it does not find perfectly matched binding sites. It is the antisense primer who can bind to the modified forward strand, and so it is extended upstream. Its extension gets to the segment from which the sense primer was derived. Any original C in that segment, which was bisulfite-modified to T, will be copied as A. And here we have a newly created reverse strand, antisense primer extension with 'modified' genotype, where the sense primer can comfortably anneal at.
Thanks for your input. It somehow made me look at the issue differently.
javier
yes it can be confusing, but with a few little drawings and scribblings you will find the answer, looks like you have already :-)
Yes, that's right, I think I got it now. In fact, what is important to understand is that primers are design to amplify only one strand from the converted DNA and that the sense primer does not anneal anywhere in the first cycle.
Could it be useful to try as well a couple of primers designed to amplify the other strand ? I guess that, depending on the sequence, the efficiency of the PCR can vary from one strand to another, am I right ??
Thanks, it is great to find some support for my first steps in the methylation world
amplifying on the other strand works just as well, and we have tested this because we feared there may be strand-biased DNA methylation occurring at our site of interest, it was not the case.
You run into problems if your primer sets were designed for traditional PCR ie: to each strand, the efficency drops to nothing! funny that!
Nick