Rapid A3 haplotype identification method. Upper panel: schematic representation of the part of the human EPCR gene exon 4 containing G 6936, which identifies the A3 haplotype. The 6936 mutagen primer contains 2 foreign nucleotides at positions n^–4 and n^–3 from the 3′ end (indicated by asterisks) in order to create a restriction site for the endonuclease PstI when the amplified fragment contains an A at position 6936, which corresponds to haplotype A1 or A2; the amplified fragment containing a G, which corresponds to haplotype A3, remains undigested. After genomic amplification using this primer and the 7190Rv primer, the PCR-amplified fragment contains a PstI site (CTGCA/G; underlined) when nucleotide 6936 is an A. In the amplified fragment, the part corresponding to the primer is shown in lower letters. Lower panel: 2% agarose gel electrophoresis of digested PCR products obtained using 6936 mutagen and 7190Rv primers. Lanes i, ii, iii: subjects homozygous for an A at position 6936 (A1/A1, A2/A2, and A1/A2, respectively); a restriction site for PstI was created, allowing the amplified fragment to be completely digested into 2 fragments of 254 and 36 bp (the latter is not visible on the gel). Lane iv: subject homozygous foraGat position 6936 (A3/A3): no PstI restriction site is available, and the fragment remains undigested at 290 bp. Lanes v-vi: subjects heterozygous A/G at position 6936 (A1/A3 or A2/A3; ie, A3 “heterozygotes”): both patterns are visible, corresponding to the undigested (290 bp) and digested (254 bp) amplified fragments. Lane vii: undigested PCR-amplified fragment.