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The molecular analysis is
carried out by using a multiplexed SnapShot protocol. For this purpose, the
PCR amplification and cleanup is followed by cloning to confirm allelic
identities. After that, DNA sequencing of about 50 ind is carried out to
identify SNPs. Then multiple SNP primers are designed that would end right
before the SNP sites. The SNP genotyping protocol is a single base pair
extension protocol, meaning the primers are extended for only 1 base,
covering the SNP site, and the reaction is terminated. So you end up getting
products only 1 bp larger than the primer. Multiplexed SNapShot protocol
requires the use of primers of different lengths that can all be combined in
a single test tube. The length differences in primers may be obtained by
adding poly T or poly GACT tails..
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