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 (HIV-1) reverse transcriptase (RT) gene results in decreased sensitivity to zidovudine (ZDV). In order to follow changes in codon 215 mutant (MUT) and wild-type (WT) populations in the plasma of patients during therapy, two polymerase chain reaction (PCR) procedures were investigated. The first was a nested, selective PCR, wherein a first round with viral-specific primers was followed by a second round with allele-specific primers. Although the procedure is relatively sensitive, some samples in the first round of PCR could not be amplified. In mixing experiments, mispriming of the MUT primer made relative determination of quantities subjective and difficult. Differential hybridization of PCR product 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 amount of PCR product analyzed. Differential hybridization was linear and reproducible over several logs of MUT:WT ratios, and determination of a 1:100 ratio of MUT:WT was readily achieved. When applied to longitudinal samples from three patients, dramatic changes in each population were readily apparent. These changes were evaluated with regard to viral load.
Eastman
P S
PS
Chiron Corporation, Emeryville, CA 94608-2916, USA.
Urdea
M
M
Besemer
D
D
Stempien
M
M
Kolberg
J
J
D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded; D016049:Didanosine / Q000627:therapeutic use; D004352:Drug Resistance, Microbial / Q000235:genetics; D004359:Drug Therapy, Combination; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000235:genetics / Q000821:virology; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D015215:Zidovudine / Q000627:therapeutic use
D003062:Codon; D017931:DNA Primers; D015342:DNA Probes; D004277:DNA, Single-Stranded; D012367:RNA, Viral; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016049:Didanosine