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 (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.
Blömeke
B
B
Department of Dermatology, University Hospital, Pauwelsstrasse 30, RWTH Aachen, 52074, Germany. B.BLOEMEKE@RWTH-AACHEN.DE
Sieben
S
S
Spötter
D
D
Landt
O
O
Merk
H F
HF
D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length
D005456:Fluorescent Dyes; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human