DNA repeat screening in GC-rich promoterregion - (Dec/06/2004 )

Hi,
I found a 9bp repeat (3-7x) in a promoterregion. The repeat itself is GC-rich as is the surrounding sequence. Because of the GC-content I had to design primers which yield a product of 420bp, a bit too long for a 9 bp repeat. Nested PCR's yielded additional interfering PCR products (I tried different annealing temp). I have tried several primersets to amplify the repeat but I can't get rid of the heteroduplexes also. I have tried different urea gels with urea and/or formamide loading buffers. Does anybody have an idea how to solve it, or should I use labeled primers and Gene Scan to screen for this repeat (of course the products need to be separated anyway)

Henkjan

hi
i am also working on same kind of thing , since i have optimized my pcr for repeat region of my gene , now i ve to run gene scan ,but i have no idea about this concept , technical thing i can do but i want to know its working principle and chemistry, wiil u please help me with that,
thnks
kavs


Hi,
I found a 9bp repeat (3-7x) in a promoterregion. The repeat itself is GC-rich as is the surrounding sequence. Because of the GC-content I had to design primers which yield a product of 420bp, a bit too long for a 9 bp repeat. Nested PCR's yielded additional interfering PCR products (I tried different annealing temp). I have tried several primersets to amplify the repeat but I can't get rid of the heteroduplexes also. I have tried different urea gels with urea and/or formamide loading buffers. Does anybody have an idea how to solve it, or should I use labeled primers and Gene Scan to screen for this repeat (of course the products need to be separated anyway)

Henkjan
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