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).
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).
Schlüter
Bernhard
B
Institute of Clinical Chemistry and Laboratory Medicine, University of Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. schlber@uni-muenster.de
Erren
Michael
M
Schotte
Heiko
H
Junker
Ralf
R
Rust
Stephan
S
Assmann
Gerd
G
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*
D014409:Tumor Necrosis Factor-alpha