Validation of Four Multiplex SNP Panels for Forensic DNA Testing: An Assessment of the Sensitivity and Reproducibility of the SNaPshot Primer Extension Assay

Orchid Cellmark provided four proprietary multiplex SNP primer panels, each of which has been developed to identify 10-12 SNPs using a modified version of the SNaPshot Mutliplex protocol. The commercially available STR typing kits routinely used in forensic testing require an input of between 0.5 to 2ng of DNA. Orchid Cellmark has suggested using 2ng of DNA with each of their multiplex SNP primer panels. However, preliminary data has indicated that as little as 100pg of DNA can yield results with many of the SNP markers. Several methods of SNP detection exist. This project relied on the use of multiplex SNP extension primers in conjunction with the ABI SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA) to identify SNPs in the nuclear genome. Analysis of 50 or more SNPs would be very laborious if single-tube polymerase chain reaction (PCR) was used for sample testing. Multiplexing SNP primers to include 10-12 per reaction tube will increase the throughput of SNP analysis. The cost of analysis can also be reduced using multiplexes since the amount of reagents per SNP is decreased. This project determined if the SNaPshot extension assay used in conjunction with the Orchid Cellmark multiplex panels could accurately detect SNPs at quantities less than 2ng DNA on a capillary electrophoresis (CE) platform.