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Microsatellites size polymorphism false-positive peaks

Sat Jun 10, 2006 2:38 pm

I developed 4 primers-pairs for amplification of 4 SSR-loci of rodents. For each of these primers-pairs the forward primer was fluorescently labeled (CY5), and polymorphism test was carried out in ALF machine.

The expected alleles size is about 120-220bp, and I can clearly see 1-2 peaks with the expected size on ALF. The problem is that I get some extra large peaks of 70-140bp that sometimes overlap with the expected bands. This is NOT a stuttering problem.

I just don't know how to remove these bands.

I saw one clear band on gel electrophoresis for each microsatellite PCR product (with Ethidium Bromide).I sequenced the bands to make sure I get what I want. Thus, I think the extra peaks on the ALF are false-positive peaks. I also excluded the option of multiple loci by using "In-Silico PCR" (virtual PCR). I tried to increase PCR stringency but I get only small improvement (annealing temp, Mg).

Thanks in advanced for your responses

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