Hi Everyone,
I am working on tetra-ARMS-PCR to genotype the rs6387. The size of amplicons is: control 610bp, A allele 281bp and G allele 382bp.
The PCR is showing a perfect amplification with clear difference among all three genotypes (AA, GG, GA) but the Sanger sequencing results of AA genotype (according to ARMS-PCR) are showing a sharp peak of G instead of A . However, the sequencing result of GA genotype is showing peaks for both G and A.
I repeated the sequencing with different sample (with AA genotype according to PCR results) but the sequencing results are same.
In gel pictures (attached with question), all three genotypes are clearly distinct.
please help me trouble shoot this problem.
Thanks,
Misbah
I think the sequencing results are more reliable then PCR gel pictures...Is GA appears for all samples of AA genotypes??
No. GA sample sequencing results are ok. But problem is only with GG. Even AA results are correct and in accordance to gel picture.
Hi,
The sharpness of peak at chromatogram may be related to quality of nucleotide and this depend on your PCR product which itself related to extracted DNS , you can try more than one sample to check the result.
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