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.
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.
Rasmussen
Henrik
H
Research Institute of Biological Psychiatry, H:S Sct. Hans Hospital, 2 Boserupvej, 4000 Roskilde, Denmark.
Werge
Thomas
T
D000483:Alleles; D020029:Base Pairing; D004247:DNA / Q000032:analysis / Q000235:genetics; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics*
D019713:Receptors, CCR5; D004247:DNA