In Sanger sequencing, what are the possible reasons a particular point mutation to become homozygous from the Forward primer while being the same mutational point a heterozygous in the chromatogram generated from the Reverse primer?
Sequencing was done at Macrogen, their suggestion was to go for a resequencing.
if the template is mixed pcr amplimer with both alleles and the reverse sequencing primer is annealing to both allelic products it will show both changes. If the forward primer has its 3' end sitting over a polymorphism then only the perfect homology sequence will anneal properly so you will sequence the strand and the allele which has the perfect homology but the 3' end of the forward primer will not anneal over the other base of the polymorphism so there will be no sequence
Dear Herath,
do you have a quality score for the reverse sequence?
Again posting the sequence would be helpful.
Hi
as Paul described, I think you have two SNP in perfect LD (linkage disequilibrium). maybe you should try to see on the UCSC if your target and primers sequences show some variations (additionnal tracks will show you so). personally after having designed primers, I test them for in silico PCR and avoid presence of variation in on the genomic.
fred
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