Identification of N-acetyltransferase 2 genotypes by continuous monitoring of fluorogenic hybridization probes.
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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ABSTRACT: Polymorphism in N-acetyltransferases NAT1 and NAT2 may contribute to differences in cancer susceptibility of subjects exposed to alkylating compounds. We developed a robust method for simultaneous determination of these NAT polymorphisms: Reverse line blot (RLB) hybridization, based on PCR followed by allele-specific oligo hybridization. On a membrane, allele-specific oligonucleotide probes of the NAT genes (NAT1*4, *3, *10, *11 and NAT2*4, *5, *6, *7, *12) were applied in lines. After separate amplification of the NAT genes, simultaneous hybridization of these products in lines perpendicular to the lines with oligonucleotide probes was performed, followed by nonradioactive detection. This resulted in hybridization patterns, representing the NAT genotype of an individual. RLB hybridizations were conducted on DNA from 240 Dutch Caucasian participants in an ongoing case-control study on colorectal adenoma (including 126 polyp-free control subjects). Results were in complete agreement with those obtained by commonly used methods, i.e., allele-specific PCR and PCR-RFLP. Allele-frequencies in the polyp-free control group were similar to those described in the literature. RLB hybridization is, however, considerably faster and cheaper than the common assays. Moreover, expansion with allelic variants of other genes is relatively easy, which makes RLB hybridization very useful for multiplex analysis of numerous polymorphisms in epidemiological studies.Analytical Biochemistry 11/2000; · 2.31 Impact Factor
- Analytical Biochemistry 02/2001; 288(1):106-8. · 2.31 Impact Factor
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ABSTRACT: We have developed a new method for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The technique involves PCR amplification of genomic DNA with two tailed allele-specific primers that introduce priming sites for universal energy-transfer-labeled primers. The output of red and green light is conveniently scored using a fluorescence plate reader. The new method, which was validated on nine model SNPs, is well suited for high-throughput, automated genotyping because it requires only one reaction per SNP, it is performed in a single tube with no post-PCR handling, the same energy-transfer-labeled primers are used for all analyses, and the instrumentation is inexpensive. Possible applications include multiple-candidate gene analysis, genomewide scans, and medical diagnostics.Genome Research 02/2001; 11(1):163-9. · 13.85 Impact Factor