Definition:

An allelic discrimination (AD) assay is a multiplexed (more than one primer/probe pair per reaction), end-point (data is collected at the end of the PCR process) assay that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the single-nucleic polymorphism (SNP) site in a target template sequence. The actual quantity of target sequence is not determined.

An AD assay involves performing:

1.a pre-read run: to determine the baseline fluorescence

2.an amplification run: to generate real-time PCR data

3. a post-read run: to subtract the baseline fluorescence determined during the pre-read run.

DESIGNING AN AD EXPERIMENT

1. Design the experiment: a) order the TaqMan Universal PCR Master Mix (PN 4324018); b) Select and order the probes and primers.

2. Extract the DNA from samples

3. Prepare the reaction mix. The final reaction volume in each well is 25 ul.

Perform the Pre-Read Run