CE sequencing and primers

[molesgirl]

Hi All,

I am currently working on a CE sequencing assay. I am having trouble with one of my primer sets. It is flanking the exon but the exon has a 98% homologous psuedogene. The area of difference sits about halfway through the exon. I was going to try primers that specifically anneal to that area in order to limit the amount of psuedogene that keeps popping up but I remember hearing that the area that the primer anneals to should be disregarded when it comes to analysis. I remember this from a colleague designing a clinical sequencing assay, he had said you cannot have a primer anneal in an area of interest. Is this true and if so why is that? Does it have something to do with error rate of a primer annealing? I'm brand new to this so any help would be greatly appreciated!! Thanks!

[canalon]

or would that be because if your primer anneals in the area of interest, you wil never see that interesting sequence (at best only that of the primer) and you are losing interesting information.
I am not familiar with what you call CE sequencing, but I know that if i want to study difference in sequences, my primers must be confined out of the variable regions, because:
- annealing, as in if the sequence change, the annealing will be modified and I might not get as good a result as I want
- sequence, I want to have as much of the variation possible between the conserved regions, if I put my primers in the variable region, there is so much that I lose.