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

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

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 (C481T), NAT2*6A (G590A), and NAT2*7A (G857A)....

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Bibliographic Details
Published in:Analytical biochemistry Vol. 275; no. 1; pp. 93 - 97
Main Authors: Blömeke, Brunhilde, Sieben, Sonja, Spötter, Dorette, Landt, Olfert, Merk, Hans F.
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-11-1999
Subjects:
Arylamine N-Acetyltransferase - genetics
Arylamine N-Acetyltransferase - isolation & purification
Fluorescent Dyes
Genotype
Humans
Polymerase Chain Reaction - methods
Polymorphism, Genetic
Polymorphism, Restriction Fragment Length
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Summary: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 (C481T), NAT2*6A (G590A), and NAT2*7A (G857A). 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 (Tm) 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.
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ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1999.4288