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bisulfite modification of dna for pcr


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#1 kant0008

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Posted 08 March 2004 - 09:09 PM

Hi,
I have 2 questions for anyone who's done bisulfite pcrs before. Firstly, does anyone know if plasmids can be/ are easy to bisulfite-modify?
Secondly, when designing bisulfite primers, do you need to look at both strands, as in normal pcr- one primer binds to top strand, other to bottom, then they "swap" to each other's products, or do you only look at one strand for both primers? I probably am really confused, but I just can't understand how can bisulfite pcr happen if the 2 modified strands are no longer complimentary. Please help!
Thanks heaps

#2 pcrman

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Posted 10 March 2004 - 06:03 AM

does anyone know if plasmids can be/ are easy to bisulfite-modify?


Should be the same as genomic DNA

Secondly, when designing bisulfite primers, do you need to look at both strands, as in normal pcr- one primer binds to top strand, other to bottom, then they "swap" to each other's products, or do you only look at one strand for both primers?


You are right, after bisulfite modification, two strands are no longer complementary. Usually we assume that both strands are symmetrically methylated so only one strand is mapped for methylation which means you can pick primer from any strand, but usually the sense strand is used.

Hope it helps.




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