We are trying to test the efficiency of pairing of a primer to a region of mouse genome. We are thinking of digesting genomic DNA before pairing and wonder whether the longer or the shorter genomic DNA fragments interfere with the specific pairing at various effects, such as by non-specific and partial pairing.
If you are doing PCR I don't expect the digestion would make any difference, but if you are up to something unorthodox, the only consideration I can come up with is that the shorter fragments will be faster to reanneal after denaturation. That said, a mammalian genomeis so complex the reannealing would still take hours versus a second or so for the primer (if it is in concentration of 0.1 uM or higher, that is), so once again it is probably irrelevant for all practical purposes.
Thank you, Mikhail. What I am trying to do is to synthesize my target DNA linearly with just one primer. Ideally, I wish 100% of template molecules are used but apparently this is impossible. However, I hope I can get as high as 80% of molecules via multiple rounds of linear synthesis, of course without destroying the double-stranded specific products.
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