A trio of human molecular genetics PCR assays

Abstract

This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional PCR, b) perform PCR - Restriction Fragment Polymorphism (PCR-RFLP) analysis to genotype a Single Nucleotide Polymorphism (SNP) of the TAS2R38 gene on human chromosome 7 and c) perform duplex Allele Specific Primer-PCR (ASP-PCR) to genotype SNPs of two enzyme-encoding genes in a single biochemical pathway on human chromosomes 4 and 12. All PCR reactions have been optimized to use a single easily purified sample of the students' own DNA and run under a single thermal cycler program using inexpensive reagents to produce robust and clearly interpretable results on a single agarose gel. As presented here, the lab occupies two lab periods of 2 h, 40 min each: DNA purification followed by PCR reactions set-up on Day 1 and enzyme digestion of the PCR-RFLP and agarose gel analysis on Day 2.