title: Comparison study for genotyping of a single-nucleotide polymorphism in the tumor necrosis factor promoter gene.
abstract: A variety of methods that address the detection of single-nucleotide polymorphism (SNP) have been used in molecular diagnostics. The allele-specific polymerase chain reaction (ASPCR) has been one of the most extensively studied, including its application in the tumor necrosis factor (TNF)(-308) genotyping. Many studies have demonstrated that the ASPCR sensitivity and specificity depends on various PCR parameters, with mismatches occurring to a degree of 4%. The purpose of our study was to evaluate a comparison of genotyping of the TNF(-308) using an ASPCR and automated sequencing (ASEQ). In a total of 204 DNA samples, their duplicate examination by the ASPCR and ASEQ revealed concordant results in 96.5% and mismatches in 3.5% genotypes. Depending on the target TNF(-308G/G), TNF(-308G/A) , TNF(-308A/A) sequences, this translated into decreased ASPCR sensitivity to a degree of 98.6%, 94.2%, 60.0%, specificity 94.7%, 97.4%, 100.0%, positive predictive values 97.9%, 92.5%, 100.0%, and negative predictive values 96.4%, 98.0%, 99.0%, respectively. Based on these results, we found ASEQ to be more accurate than ASPCR for the TNF(-308) genotyping. By eliminating the need of empirical determination of appropriate PCR conditions for each studied sequence, ASEQ provides a sensitive and reproducible quality-control benchmark for other SNP assays.
authors.affiliation: Department of Hematology, Medical University of Lodz, Poland.
authors.forename: Przemysław
authors.initials: P
authors.lastname: Juszczynski
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Woszczek
authors.forename: Maciej
authors.initials: M
authors.lastname: Borowiec
authors.forename: Marek
authors.initials: M
authors.lastname: Kowalski
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Robak
authors.forename: Przemysław
authors.initials: P
authors.lastname: Biliński
authors.forename: Gilles
authors.initials: G
authors.lastname: Salles
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Warzocha
mesh_terms: D001331:Automation; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D014409:Tumor Necrosis Factor-alpha