A closed-tube assay for genotyping of the 32-bp deletion polymorphism in the chemokine receptor 5 (CCR5) gene: dissociation analysis of amplified fragments of DNA

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Abstract

We have developed a closed-tube assay for determination of the chemokine receptor type 5 (CCR5) 32-bp deletion allele, which protects against infections with HIV and modulates susceptibility to a variety of inflammatory diseases. This assay utilizes dissociation analysis of amplified products in the presence of Sybr Green I for allele discrimination. After having established robust conditions for the assay, we used it to genotype 590 unknown DNA samples. A blinded comparison with a procedure based upon agarose gel electrophoresis of amplified material revealed complete concordance between the two procedures. Our closed-tube assay is inexpensive and easy to carry out. Furthermore, it reduces or eliminates the risk of carry-over contamination with previously amplified products. The insights gained in this study can be applied to develop assays for genotyping of other insertion/deletion polymorphisms based upon differences in T(m) of allele-specific amplicons.
OriginalsprogEngelsk
TidsskriftMolecular and Cellular Probes
Vol/bind21
Udgave nummer1
Sider (fra-til)8-11
Antal sider4
ISSN0890-8508
DOI
StatusUdgivet - 2007

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