TY - JOUR
T1 - A closed-tube assay for genotyping of the 32-bp deletion polymorphism in the chemokine receptor 5 (CCR5) gene
T2 - dissociation analysis of amplified fragments of DNA
AU - Rasmussen, Henrik Berg
AU - Werge, Thomas
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
U2 - http://dx.doi.org/10.1016/j.mcp.2006.05.003
DO - http://dx.doi.org/10.1016/j.mcp.2006.05.003
M3 - Journal article
SN - 0890-8508
VL - 21
SP - 8
EP - 11
JO - Molecular and Cellular Probes
JF - Molecular and Cellular Probes
IS - 1
ER -
