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Pyrosequencing

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Pyrosequencing

Postby kk » Wed Jan 23, 2013 3:08 pm

Hello, I would like to sequence a gene region with a couple of mutation hot spots nearby each other, and I have a few questions. I guess after PCR amplification of the gene region of interest, I should use a sequencing primer that anneals to a region that has no mutation hot spots. The I generate my dispensation order, and when I reach a mutation hot spot, I add two dispensations - one for the wild type and one for the mutant nucleotide. Question1: how come that these hot spots always change from e.g. G to C and not G to any nucleotide? I mean should I include all 4 nucleotides in the dispensation order or just the one identified by the literature. Question2: how does homozygosity or heterozygosity affect the pyrogram, I mean I will have total DNA isolated from a tumor tissue, which ideally in all cells=all DNA molecules the mutation, so in theory all DNA carries the mutant nucleotide, but what if only one allele is mutant, how does it show on the pyrogram, how should I adjust the dispensation order then? tx so much
kk
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Postby kk » Fri Feb 01, 2013 9:04 am

I got the answer to question one, cosmic mutational database answered my question (it is not certain, that a C for example always changes to G, it could be T or A at a much lower frequency). But then: Should I include those rare variants too in the dispensation order?

Another thing, which mode should I use to design such assays: SNP, AQ, SQA?
kk
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Postby kk » Tue Feb 12, 2013 2:42 pm

In AQ mode one can design point mutation detecting dispensation orders and peak height ration will tell the prevalence of mutation in a wild type background.
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