HFE C282Y mutation detection assay.
Lanes 1 and 14 contain 50 bp marker as indicated at the left of the figure. Lanes 2-7 contain samples as indicated above the gel. All reactions were performed with the original Feder et al (1996) primers. Lanes 8-13 contain the samples indicated above the gel. All reactions were performed with the original Feder et al2 forward primer and the Jeffrey et al4 reverse primer. Known polymorphic samples are indicated (PM 1986, PM 2147) and were prepared in the Molecular Diagnostic Lab of the University of Alberta as described in Somerville et al.5 All other DNA samples were obtained from either blood or buccal cells and were prepared in the MSU DNA Diagnostic Laboratory by a standard alkaline lysis procedure. Each PCR reaction contains 12.5 pmoles each primer, 1× Rediload (Research Genetics), 10 mmol/L dNTPs, 1× GeneAmp PCR buffer (Perkin-Elmer: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% w/v gelatin), and 0.25 U Taq DNA polymerase in a total reaction volume of 25 μL. Conditions for amplification: 95°C, 5′; 30 cycles of 94°C, 30"; 63°C, 30"; 72°C 30"; hold at 4°C. All PCR reactions were performed in a Perkin-Elmer 9600 thermocycler. PCR products (390 bp) are digested with RsaI to give 250 bp + 140 bp for a normal result, or 250 bp + 140 bp + 111 bp + 29 bp for a C282Y carrier, or 250 bp + 111 bp + 29 bp for a mutant C282Y homozygote. Restriction digestion products are run on a 3% Nu-Sieve 3:1 (FMC BioProducts) agarose gel in 0.5× TBE.
