Identification of N-Acetyltransferase 2 Genotypes by Continuous Monitoring of Fluorogenic Hybridization Probes

✍ Scribed by Brunhilde Blömeke; Sonja Sieben; Dorette Spötter; Olfert Landt; Hans F. Merk

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 229 KB
• Volume: 275
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4288

✦ Synopsis

Three polymorphic sites in the N-acetyltransferase 2 (NAT2) gene were detected using rapid cycle DNA amplification with allele-specific fluorescent probes and melting curve analysis. Two fluorogenic adjacent hybridization probes were designed to NAT2*5A (C 481 T), NAT2*6A (G 590 A), and NAT2*7A (G 857 A). During amplification, probe hybridization is observed as fluorescence resonance energy transfer. The fluorescence increases every cycle as the product accumulates during amplification. A single base mismatch resulted in a melting temperature shift (T m ) of 5 to 6°C, allowing for the easy distinction of a wild-type allele from the mutant allele. The protocol is rapid, requiring 40 min for the completion of 45 cycles including the melting curves. It is also a simple and flexible method, since DNA templates prepared from different sources, including DNA from serum and paraffin-embedded tissue sections, could be used without adverse effects. Fluorescence genotyping of all three polymorphisms in a total of 155 DNA samples correlated perfectly with our previously validated genotyping by restriction enzyme digestion (PCR-RFLP). This new facile approach allows for the easy detection of NAT2 polymorphisms in hundreds of samples in only a day.