Identification of N-acetyltransferase 2 genotypes by continuous monitoring of fluorogenic hybridization probes.

Department of Dermatology, University Hospital, Pauwelsstrasse 30, RWTH Aachen, 52074, Germany.
Analytical Biochemistry (Impact Factor: 2.58). 12/1999; 275(1):93-7. DOI: 10.1006/abio.1999.4288
Source: PubMed

ABSTRACT 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 degrees 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.

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