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Design and Implementation of an Undergraduate PCR‐RFLP Genotyping Laboratory for an HIV Susceptibility Gene
Author(s) -
Dinh Nam,
Waikel Rebekah L
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a975-b
Subject(s) - genotyping , restriction fragment length polymorphism , biology , restriction enzyme , genotype , genetics , allele , restriction site , gene , polymerase chain reaction , microbiology and biotechnology , base pair , virology
We have designed a novel Chemokine Receptor 5 (CCR5) PCR‐RFLP genotyping laboratory to help students learn molecular biology concepts, techniques and applications using the contemporary topic of HIV/AIDS. CCR5 is a co‐receptor for HIV and is required for the entry of HIV‐1 into macrophages and some T‐cells. The co‐receptor was in part identified through genetic studies of high risk individuals that were resistant to HIV infection. Resistance is due to a 32 base pair deletion (Δ32), which generates a truncated CCR5 protein that is not expressed on the cell surface. CCR5 Δ32 homozygous individuals are resistant to most strains of HIV‐1. Heterozygous individuals demonstrate a delayed progression of AIDS development. PCR primers were designed to amplify 473 base pairs of the wild‐type CCR5 allele containing the region of the 32 base pair deletion. Since a 32 base pair difference in band size on an agarose gel is difficult to image, we added a restriction digest step that would yield a differential number of bands dependent of genotype. The restriction enzyme Apo I cuts twice in the 473 base pair wild‐type PCR product and only one time in the Δ32 mutant PCR product. Homozygous wild‐type individual exhibit three bands (211 bp, 153 bp, and 99 bp), whereas homozygous Δ32 individuals exhibit two bands (252 bp and 211 bp). Heterozygous individuals display four bands (252 bp, 211 bp, 153 bp, and 99 bp).