Dear all,
I have submitted a forward and reverse Sanger sequencing reaction for multiple PCR products. The goal of the sequencing reactions was to analyze single nucleotide polymorphisms to determine the parent-of-origin expression levels of several genes (maternally expressed/paternally expressed/biallelically expressed). However, the forward and reverse sequencing reactions give different parent-of-origin expression ratios (maternal nucleotide peak height divided by paternal nucleotide peak height), while the exact same PCR product was sequenced.
Does anyone know how this is possible?
I agree with Artur that electropherograms are not quantitative.If this is a result from a single primer pair then there may be a possibility of one primer sitting very near or over a polymorphism at its 3 ' end which would mean that one allele amplifies and sequences less efficiently than the other
I agree with Paul Rutland, the most possibility is missing an allele.
If you are interested in the effect that adjacent or surrounding bases have on a base intensity in sequencing there was a very old paper looking into this in Biotechniques, 21, 694-699, October 1996
but I am certain that enzymes,dideoxy concentartions and everything about sequencing kits has improved enormously with respect to unifoem peak heights since then
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