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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- "These methods can be convenient, but require an additional step for end point detection. Analytical methods that reduce sample processing to a single-step procedure were also developed, including nick-translation PCR (17), hybridization with FRET probes (18), melting curves analysis (19–22), fluorogenic allele-specific PCR (23), universal FRET reagents in a single-step Invader assay (24), universal TaqMan probes (25) and others. However, several of these methods cannot deal with detection of a low concentration of mutant target in the presence of a large excess of wild-type DNA that is often found in clinical specimens. "
ABSTRACT: Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5'-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to approximately 0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.Nucleic Acids Research 02/2007; 35(19):e131. DOI:10.1093/nar/gkm809 · 9.11 Impact Factor
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- "Promoter fragments containing more than two SNPs were genotyped by direct sequencing. All other SNPs were genotyped with the LightCycler™ assay (Roche, Mannheim, Germany) based on hybridization of probes labeled by two different dyes allowing Fluorescence Resonance Energy Transfer (FRET) . A genotyping quality control was performed by introducing duplicates in the PCR plates and by genotyping all individuals twice. "
ABSTRACT: Cocaine and amphetamine regulated transcript (CART) is an anorectic neuropeptide located principally in hypothalamus. CART has been shown to be involved in control of feeding behavior, but a direct relationship with obesity has not been established. The aim of this study was to evaluate the effect of polymorphisms within the CART gene with regards to a possible association with obesity in a Caucasian population. Screening of the entire gene as well as a 3.7 kb region of 5' upstream sequence revealed 31 SNPs and 3 rare variants; 14 of which were subsequently genotyped in 292 French morbidly obese subjects and 368 controls. Haplotype analysis suggested an association with obesity which was found to be mainly due to SNP-3608T>C (rs7379701) (p = 0.009). Genotyping additional cases and controls also of European Caucasian origin supported further this possible association between the CART SNP -3608T>C T allele and obesity (global p-value = 0.0005). Functional studies also suggested that the SNP -3608T>C could modulate nuclear protein binding. CART SNP -3608T>C may possibly contribute to the genetic risk for obesity in the Caucasian population. However confirmation of the importance of the role of the CART gene in energy homeostasis and obesity will require investigation and replication in further populations.BMC Genetics 04/2005; 6:19. DOI:10.1186/1471-2156-6-19 · 2.40 Impact Factor
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- "It should be noted that the study presented here is preliminary and its statistical power is limited due to the relatively small number of samples analysed, particularly the study of NAT2 genotype and risk of AD. Therefore much larger case-control investigations, using more highly automated detection methods such as the LightCycler real-time PCR methods of Blömeke et al.  and Wikman et al.  will help to validate the results shown here. In order to detect a 1.5 fold increased risk of AD associated with the NAT1*10 allele, approximately 650 cases and 650 controls would need to be studied to give 80% power of achieving significance at the 5% level. "
ABSTRACT: Alzheimer's disease is multifactorial, having environmental, toxicological and genetic risk factors. Impaired folate and homocysteine metabolism has been hypothesised to increase risk. In addition to its xenobiotic-metabolising capacity, human arylamine N-acetyltransferase type-1 (NAT1) acetylates the folate catabolite para-aminobenzoylglutamate and is implicated in folate metabolism. The purpose of this study was to determine whether polymorphisms in the human NAT genes influence susceptibility to Alzheimer's disease. Elderly individuals with and without Alzheimer's disease were genotyped at the polymorphic NAT1 (147 cases; 111 controls) and NAT2 (45 cases; 63 controls) loci by polymerase chain reaction-restriction fragment length polymorphism, and the genotype and allele frequencies were compared using the chi-squared test. Although a trend towards fast NAT2 acetylator-associated Alzheimer's disease susceptibility was indicated and the NAT1*10/1*10 genotype was observed only in cases of Alzheimer's disease (6/147, 4.1%), no significant difference in the frequency of NAT2 (p = 0.835) or NAT1 (p = 0.371) genotypes was observed between cases and controls. In addition, a novel NAT1 variant, NAT1*11B, was identified. These results suggest that genetic polymorphisms in NAT1 and NAT2 do not influence susceptibility to Alzheimer's disease, although the increase in frequency of the NAT1*10 allele in Alzheimer's disease is worthy of further investigation. Due to its similarity with the NAT1*11A allele, NAT1*11B is likely to encode an enzyme with reduced NAT1 activity.BMC Medical Genetics 04/2004; 5(1):6. DOI:10.1186/1471-2350-5-6 · 2.08 Impact Factor