COMPARISON OF SELECTIVE POLYMERASE CHAIN-REACTION PRIMERS AND DIFFERENTIAL PROBE HYBRIDIZATION OF POLYMERASE CHAIN-REACTION PRODUCTS FOR DETERMINATION OF RELATIVE AMOUNTS OF CODON-215 MUTANT AND WILD-TYPE HIV-1 POPULATIONS

A mutation at codon 215 of the human immunodeficiency virus type 1 (HI V-1) reverse transcriptase (RT) gene results in decreased sensitivity to zidovudine (ZDV), In order to follow changes in codon 215 mutant (M UT) and wild-type (WT) populations in the plasma of patients during th erapy, two polymerase chain reaction (PCR) procedures were investigate d. The first was a nested, selective PCR, wherein a first round with v iral-specific primers was followed by a second round with allele-speci fic primers. Although the procedure is relatively sensitive, some samp les in the first round of PCR could not be amplified. In mixing experi ments, mispriming of the MUT primer made relative determination of qua ntities subjective and difficult, Differential hybridization of PCR pr oduct with probes specific for codon 215 MUT or WT sequences was also Investigated. A probe directed to a highly conserved region of the RT gene in the amplified PCR product was used to determine the total amou nt of PCR product analyzed. Differential hybridization was linear and reproducible over several logs of M UT: WT ratios, and determination o f a 1:100 ratio of MUT:WT was readily achieved. When applied to longit udinal samples from three patients, dramatic changes in each populatio n were readily apparent. These changes were evaluated with regard to v iral load.