SNaPshot Nuclear SNP Detection
q PCR amplification and cleanup

q Cloning to confirm allelic identities

q DNA sequencing of  50 individuals to identify SNPs
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q Design of  SNP primers using SBE Primer software

q SNP genotyping using multiplexed ABI SNaPShotTM protocol (7-10 SNP sites per gene;       3 genes)
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q SNP detection by GeneMapper software
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F:\ASLO Orlando\Snapshot_results_copy.jpggmr0289fig1
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..
The results will look like this figure. The individual SNP sites are homozygote if there is only one peak and heterozygote if there are two peaks.

In this way, we are planning to finish the analysis of 7-10 SNP sites per gene for 3 genes, and I will show you some of the  results of this analysis now.