Document 2750
 DOCN  M94A2750
 TI    Quantitation of HIV-1 proviruses in peripheral blood lymphocytes by
       competitive nested PCR.
 DT    9412
 AU    Hiraishi Y; Kato S; Asakawa M; Takano T; Department of Microbiology,
       Keio University School of Medicine,; Tokyo, Japan.
 SO    Int Conf AIDS. 1994 Aug 7-12;10(1):233 (abstract no. PB0360). Unique
       Identifier : AIDSLINE ICA10/94369825
 AB    OBJECTIVE: To optimize the conditions of the competitive nested PCR
       assay which is highly sensitive for quantitation of HIV-1 DNA. METHODS:
       The target sequence was within the nef gene. The competitor DNA was
       produced by an internal deletion of the parental HIV-1 DNA. Competitor
       DNA with a known copy number was co-amplified with 1 microgram of DNA
       from peripheral blood lymphocytes of infected individuals by two
       sequential PCR using different pairs of primers. The ratio between the
       wild-type and competitor DNA products are determined by densitometric
       analysis of DNA bands on agarose gel electrophoresis. RESULTS: To ensure
       that the ratio between the wild-type and competitor DNA products be
       equal to the ratio of these DNA before amplification, the preheating and
       the polymerization steps had to be long enough. In the case of sample
       DNA, the amounts of wild-type DNA products were often gradually
       increased with the annealing temperature decreased, which was thought to
       be an effect of mismatches between primers and target DNA. Such an
       effect can be erased by using an annealing temperature at least 16
       degrees C below Tm of the primers. DISCUSSION AND CONCLUSIONS:
       Competitive nested PCR is useful for accurate quantitation of HIV-1
       nucleic acids with no need of radioisotope.
 DE    Genes, nef/GENETICS  Human  HIV Infections/*MICROBIOLOGY
       *HIV-1/GENETICS  Lymphocytes/*MICROBIOLOGY  Polymerase Chain
       Reaction/*METHODS  *Proviruses/GENETICS  Viremia/*MICROBIOLOGY  MEETING
       ABSTRACT

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