I am trying to perform a SSP-PCR of HLA genes. The reaction is optmized as it has worked with my controls. But when I try to amplify my cancer samples the reaction does not work.
Some samples do amplify, but I can't find a reason why some samples work while others don't.
For example, sample 167 worked (26ng/ul, 260/280 = 1.96, and 260/230 = 1.26), while sample 383 did not work (21ng/ul, 260/280 = 1.85, and 260/230 = 1.51).
All samples are from tumor, and the dna was extracted with phenol/ chloroform.
what proportion of samples do not work?
Is it possible that one of your primers anneals over a polymorphic base near to its 3'end so the polymerase cannot extend?
If so it might be worth using a primer further out or dropping the annealing temperature to see if some of the non working samples look any better
Paul Rutland more than 90% of my cancer samples don't work.
But when I use the same primers with my control samples it works. I think the problem might be related to the samples, and not the reaction.
I may be misunderstanding the problem but isn't this result what you expect. Different specific primers amplify different sequences so a panel of different primers will genotype the sequence but if you are only running 1 primer set then it will only pick up one sequence and is this what is happening in your control sample...the primer is specific for the control sequence but cannot work on other sequences?
Paul Rutland I have a set of 8 pairs of primers. They amplify HLA epitopes.
For example I have two pairs of primers that amplify groups HLA C-C1 or HLA C-C2. So every sample must amplify for at least one of them. But my cancer samples are not amplifying any of them.
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