![]() The resulting fragments were resolved on a 10% polyacrylamide gel. RFLP analysis was performed using either 5 U of NcoI (New England Biolabs) or 5 U of DdeI (Life Technologies) in a final buffer volume of 20 μL under mineral oil for 16 h at 37 ☌. The underlined bases in the 341RC and MS341R primers represent mismatched bases with template DNA. In the DdeI method, the same Nat-Hu14 primer was used together with another modified mutagenesis primer, MS341R (5′-GAC AAT GTA ATT CCT GCC CTC A-3′). For our NcoI method, the Nat-Hu14 primer ( 8) was used with a new mutagenesis primer, 341RC (5′-C GAC AAT GTA ATT CCT GCC GTC C-3′). Amplification was performed for 30 cycles in a thermocycler (Hybaid Express), with the following conditions for each cycle: 30 s at 95 ☌, 1 min at 60 ☌, and 30 s at 72 ☌. 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 (Life Technologies), and a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl 2. The latter fragment was preamplified as described previously by Hickman and Sim ( 8), using their Nat-Hu14 and Nat-Hu16 primers. ![]() For both methods, a seminested PCR was performed to amplify a 360-bp DNA fragment using a 998-bp DNA fragment as a template. Two different RFLP methods were used for detection of the T 341C mutation: a previously described DdeI method ( 3), and a new complementary NcoI-RFLP. The C-to-T mutations at the 282 and 481 sites were detected with RFLP analysis of a 360-bp DNA fragment with FokI or KpnI digestions, respectively ( 3). 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.Įstablished PCR-RFLP methods were used to detect NAT2 mutations at the C 282T, T 341C, and C 481T sites. 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. 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 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. RFLP-based methods appear to be superior in genotyping studies. In some cases, sequencing of amplified DNA has been used as a direct method ( 4)( 5). 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).
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