Can you use a 10 kb amplicon for an hME/iPLEX reaction and skip PCR for multiple SNPs?
• Using a 10 kb amplicon as a starting material without PCR amplification for a hME reaction should be feasible. We worked with doing more genomic amplification studies before at Sequenom. One drawback which I will point out to you (as R&D pointed it out to me) is that they noticed a lot of false positives in when doing genomic amplification which is one of the main reasons why we use short amplicons in our present protocols. Your extend primer could anneal on a site which is not the target region and then become extended. Further, there is no quick and easy control experiment for these false binding sites. If you expected a C or T and got an A then you have this problem going on. Just to give you some warning of possible pitfalls you could encounter. As for the quantity involved, based on your system, R&D has suggested 1 ug of 10 kb DNA template to be used for the extension reaction which is pretty close to my 2.7 ug estimate I made. This number should be accomplishable where you have
