Document 1218
 DOCN  M9591218
 TI    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.
 DT    9509
 AU    Eastman PS; Urdea M; Besemer D; Stempien M; Kolberg J; Chiron
       Corporation, Emeryville, CA 94608-2916, USA.
 SO    J Acquir Immune Defic Syndr Hum Retrovirol. 1995 Jul 1;9(3):264-73.
       Unique Identifier : AIDSLINE MED/95308202
 AB    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.
 DE    Base Sequence  Codon/*GENETICS  Comparative Study
       Didanosine/THERAPEUTIC USE  Drug Resistance, Microbial/GENETICS  Drug
       Therapy, Combination  DNA Primers/CHEMISTRY  DNA Probes/CHEMISTRY  DNA,
       Single-Stranded  Genotype  Human  HIV Infections/DRUG
       THERAPY/GENETICS/VIROLOGY  HIV-1/DRUG EFFECTS/ENZYMOLOGY/*GENETICS
       Molecular Sequence Data  Nucleic Acid Hybridization  *Point Mutation
       Polymerase Chain Reaction/*METHODS  Reverse Transcriptase/*GENETICS
       RNA, Viral/*ANALYSIS  Zidovudine/THERAPEUTIC USE  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).