Genotyping the BDNF rs6265 (val66met) polymorphism by one-step amplified refractory mutation system PCR.
BACKGROUND
The brain derived neutrophic factor (BDNF), a 27 kD polypeptide, is one of the most widely expressed neurotrophins in the brain, regulating neural development and plasticity. The BDNF gene contains a functional single-nucleotide polymorphism (rs6265), which results in a valine to methionine substitution (val66met), leading to reduced mature BDNF expression. This polymorphism has been widely implicated in a host of psychiatric disorders and is a focus of many ongoing psychiatric genetic studies.


OBJECTIVE
To develop an efficient and rapid method to detect the val66met polymorphism in a one-step PCR reaction.


RESULTS
We have designed four PCR primers that amplify the BDNF gene region containing rs6265. The specificity of the four primers in a single PCR reaction amplifies two allele-specific amplicons (253 and 201 bp) and the entire region (401 bp) as an internal control, which are easily distinguished on a polyacrylamide gel. The effectiveness and efficiency of the results are validated by traditional NlaIII restriction enzyme digestion, sequencing of resulting bands and confirmation on 308 genomic DNA samples.


CONCLUSIONS
This new method describes a rapid, sensitive, cost effective and high throughput genotyping of the BDNF val66met polymorphism, ideal for large-scale genotyping studies.
Sheikh
Haroon I
HI
Molecular Genetics Unit, Department of Biology, University of Western Ontario, London, Ontario, Canada. hsheikh6@uwo.ca
Hayden
Elizabeth P
EP
Kryski
Katie R
KR
Smith
Heather J
HJ
Singh
Shiva M
SM
D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results
D019208:Brain-Derived Neurotrophic Factor
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