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Single-nucleotide polymorphisms have been detected by various methods, including allele-specific oligonucleotide hybridization (1), allele-specific amplification (2), and restriction fragment length polymorphism (RFLP) analysis (3). In some cases, sequencing of amplified DNA has been used as a direct method (4)(5). RFLP-based methods appear to be superior in genotyping studies. In fact, recently, Cascorbi and Roots (6) recommended the use of RFLP in preference to other methods for single-nucleotide polymorphism detection in the N -acetyltransferase-2 ( NAT2) gene. Some groups have used more than one method in their analyses, and when discrepancies occurred between two methods, they used DNA sequencing as a gold standard (7). In this report, we describe a comparison of RFLP methods with automated DNA sequencing for the detection of three different mutations in the NAT2 gene. Our results indicate that caution is needed in the use of automated sequencing to detect heterozygous mutations accurately at some polymorphic sites in the NAT2 gene.Established PCR-RFLP methods were used to detect NAT2 mutations at the C282T, T341C, and C481T sites. The C-to-T mutations at the 282 and 481 sites were detected with RFLP analysis of a 360-bp DNA fragment with Fok I or Kpn I digestions, respectively (3). Two different RFLP methods were used for detection of the T341C mutation: a previously described Dde I method (3), and a new complementary Nco I-RFLP. For both methods, a seminested PCR was performed to amplify a 360-bp DNA fragment using a 998-bp DNA fragment as a template. The latter fragment was preamplified as described previously by Hickman and Sim (8), using their Nat-Hu14 and Nat-Hu16 primers. The seminested PCR mixture (50 μL) contained 2 μL of 100-fold diluted 998-bp DNA, 200 μM each dNTP, 1.0 μM primers, 1.25 U of Taq DNA polymerase …

clinical chemistry

A Precaution in the Detection of Heterozygotes by Sequencing: Comparison of Automated DNA Sequencing and PCR-Restriction Fragment Length Polymorphism Methods