A Simple RFLP-Based Method for HFE Gene Multiplex Amplification and Determination of Hereditary Hemochromatosis-Causing Mutation C282Y and H63D Variant with Highly Sensitive Determination of Contamination

Table 1

Primer sequences.

PCR primers	5 label	Sequence (5- > 3)
HFE exon 2 (H63D) upper	ATTO 565	ACATGGTTAAGGCCTGTTGC
HFE exon 2 lower	—	GCCACATCTGGCTTGAAATT
HFE exon 4 (C282Y) upper	ATTO 565	CGGGCCTTGAACTACTACCC
HFE exon 4 lower	—	ACCTCCTCAGGCACTCCTCT
STR SE33 upper	Yakima Yellow	AATCTGGGCGACAAGAGTGA
STR SE33 lower	—	ACATCTCCCCTACCGCTATA
STR FGA upper	FAM	GGCTGCAGGGCATAACATTA
STR FGA lower	—	ATTCTATGACTTTGCGCTTCAGGA
FZD1 upper	ATTO 550	CTTGTCCGGCTGTTACACG
FZD1 lower	ATTO 550	CAGGATGGTGATGGTCTTGAT

PCR conditions and digestion were as follows: Qiagen multiplex PCR kit, 10 μl final reaction with 15-50 ng DNA per tube; primers 0.2 μM final each; initial denaturation at 95°C for 5 min followed by 26 cycles of annealing at 60°C for 90 sec, extension at 72°C for 30 sec, denaturation at 95°C for 30 sec with a final round at 68°C for 10 min; and digestion overnight with SexAI and BspHI (0.75 and 1.5 UI/μl final, respectively).