A microtitre format point mutation assay: application to the detection of drug resistance in human immunodeficiency virus type-1 infected patients treated with zidovudine

Abstract

An assay for the analysis of point mutations in DNA fragments amplified by the polymerase chain reaction is described. The method was applied to the analysis of mutations leading to single amino acid changes in the reverse transcriptase gene of human immunodeficiency virus type-1 that are associated with decreased sensitivity of the virus to the effects of the nucleoside analogue zidovudine. The assay uses a microtitre format allowing large numbers of clinical samples to be analysed, and is a quantitative assay giving information on the relative proportions of wild-type and mutant sequence at the point being analysed. The analysis of mutations in codons 67, 70, 215, and 219 of the RT gene in samples taken at various time points after the commencement of zidovudine therapy shows a high variability in the rate at which these mutations arise and in the proportions of wild-type and mutant sequences observed. The method we describe has wide application to the analysis of other point mutations in clinical or research studies.