Genomic DNA was extracted from leukocytes of the peripheral blood of the patients, and exons 1 to 11 of the
CYP4V2 gene were amplified by polymerase chain reaction (PCR; DNA Thermocycler 9700; Applied Biosystems, Foster City, CA). Primers were obtained according to previously published sequences.
8 PCR reactions were carried out in 50-μL reaction volumes containing 10 mM TrisHCl (pH 8.9), 50 mM KCl, 1.5 mM MgCl
2, 25 pmol each primer, 200 μM each deoxyribonucleoside triphosphate, 50 to 100 ng patient genomic DNA, and 0.7 U
Taq thermostable DNA polymerase (Promega, Madison, WI). Cycling parameters were 3 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at the melting temperature (
T m ) of the primers (52°C–62°C), and 30 seconds at 72°C, with a final 5-minute extension at 72°C. PCR products were purified using PCR cleanup columns (GFX; Amersham, Piscataway, NJ). Sequence variations were identified by automated bidirectional sequencing (BigDye Terminator, v3.1 chemistry; Applied Biosystems). An automated DNA sequencer (Model ABI PRISM 3100; Applied Biosystems) was used. Primers for sequence reactions were the same as those for the PCR reaction.
The presence of a 15-bp deletion mutation in IVS6-exon 7 (see below) was investigated in 236 healthy Chinese control subjects using a restriction endonuclease assay, or by direct sequencing. The PCR products (10 μL) were digested with 1 U of MwoI enzyme in the corresponding buffer by incubating overnight at 60°C. DNA fragments were detected by electrophoresis on 2% agarose gels.