title: The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-alpha promoter polymorphism (-308 G/A).
abstract: BACKGROUND
  The tumour necrosis factor-alpha (TNF alpha) promoter polymorphism (-308 G/A) has been shown to be associated with the susceptibility to and/or the severity of diverse diseases such as infections, autoimmunity, and malignancies. We developed a genotyping technique based on the mutagenically separated polymerase chain reaction (MS-PCR) which may be useful in the clinical risk assessment.
  
  
  METHODS
  Different length allele-specific primers and an unspecific complementary strand primer were used in a one-tube assay. At least one PCR product was generated in a single reaction obviating the need for an internal control amplification. Introduction of additional base substitutions into the allele-specific primers led to a clear-cut separation between the alleles through the reduction of cross-reactions during amplification. The only post-PCR step required was the separation of allelic PCR products by size upon agarose gel electrophoresis.
  
  
  RESULTS
  The allele frequencies in 300 German healthy Caucasians were 0.84 for TNF1 (-308 G) and 0.16 for TNF2 (-308 A) in accordance with published data obtained with the conventional RFLP method. No significant deviation from Hardy-Weinberg equilibrium was observed. The specificity of MS-PCR was confirmed by sequence-based typing.
  
  
  CONCLUSIONS
  MS-PCR is a rapid, reliable, and cost-effective technique for genotyping of the TNF alpha promoter polymorphism (-308 G/A).
authors: Institute of Clinical Chemistry and Laboratory Medicine, University of Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. schlber@uni-muenster.de
authors: Bernhard
authors: B
authors: Schlüter
authors: Michael
authors: M
authors: Erren
authors: Heiko
authors: H
authors: Schotte
authors: Ralf
authors: R
authors: Junker
authors: Stephan
authors: S
authors: Rust
authors: Gerd
authors: G
authors: Assmann
mesh_terms: D000328:Adult; D005260:Female; D005787:Gene Frequency / Q000235:genetics; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D014409:Tumor Necrosis Factor-alpha