One fluorescent based method for genotyping small numbers of SNPs is a fluorogenic 5' nuclease chemistry, referred to as the TaqMan method as it uses the same technology used for real-time or quantitative PCR, developed by Applied Biosystems, Inc. In each TaqMan 'allelic discrimination' assay contains two probes each with different reporter dyes at the 5' end, usually Fam and Vic and are allele specific.
The probes also contain a minor groove binding (MGB) molecule at the 3' end along with a quencher molecule. The MGB molecule functions to stabilize duplex formation, allowing greater differences in melting temperatures between perfect matches and mismatches. During PCR with unlabeled primers, each probe anneals specifically to complementary sequences between the PCR primer sites.
The 5' nuclease activity of Ampli Taq Gold DNA polymerase (Applied Biosystems, Inc.) can cleave only probes that hybridize to the allele specific sequence, thus separating the reporter dye from the quencher, resulting in fluorescence emission by the reporter dye. This fluorescent signal identifies the allele(s) present in the sample.
This service is supported by an Applied Biosystems model 7900 Fast real-time PCR system. Samples can be run in 96- or 384-well formats. The system performs both the PCR (and can generate real-time data for each TaqMan assay), and then reads the fluorescence in the wells, post-amplification. Large numbers of plates can be run easily by performing the PCR on external thermal-cyclers such as our two MJ tetrads, each with four-384 well blocks, or our two MJ tetrads, both with four-96 well blocks, and using the ABI 7900 for fluorescence scanning only.
Investigators can run their own assays and use the ABI 7900 for the scanning, or both PCR and scanning for a nominal fee. Or the Biopolymer/Genomics Core staff can run the assays for the investigators for a fee that covers materials and labor. We run assays in 384 well formats to keep costs down. Investigators must provide samples in 384 well trays. A calendar system is used to sign up for time on the instrument.
|384 well tray, TaqMan Genotyping; core provides reagents, excluding TaqMan assay*
||$250/384 well plate
|Use of Applied Biosystems 7900 Fast RT PCR system
* If TaqMan assays will be used repeatedly, the Core will be happy to stock those assays.