Diagnostic PCR troubleshooting - (Jul/21/2015 )

Hello...I'm attempting to genotype two closely related strains of an insect using primers I designed. They share a common gene but possess different alleles, the primers designed should be able to differentiate between the two alleles. I've approached this experiment using two PCR reactions. The first reaction isolates the common gene. After verifying the presence of this gene I dilute the PCR product 1:1000 and use 1uL of the first reaction's product as a template for the next PCR reaction with the new specific primers. I'm investigating four genes, eight alleles, and some of the diagnostic primers are specific, and some aren't. I'm not sure what the issue may be. There are a ton of details regarding protocol but I figured I'd get the ball rolling here with this. Any advice would be greatly appreciated!  

Didn't get some of the information:

First you wrote that you use a common gene and amplify it but later you have four genes? So you do this approach with four different common genes?

Each gene has only two alleles? Do you know how conserved the genes are?

Anyway shouldn't it be good enough if one or two of the primers are specific and allow a differentiation of the strains?

If not you can try to increase specificity by PCR modifications (e.g. annealing temperature, additives, primers).

And finally do you clean up the first PCR product? If not, even with dilution you might amplify e.g. unspecific amplicons, primer-dimers, genomic DNA which is still available.

Simplest is to pcr amplify the relevant region with non-specific primers, and then sequence it. Using PCR to differentiate sequences is very old-school, and difficult to get right without work (as you see). It MIGHT be worth it if you have hundreds of samples, but even then I would be doing sequencing.