Document 0858
 DOCN  M9480858
 TI    Sensitivity and application of a new method for isolating purified
       compartmentalized HIV-1 unintegrated and integrated DNA.
 DT    9410
 AU    Bush CE; Golembieski A; Donovan RM; Baxa D; Markowitz N; Saravolatz LD;
       Henry Ford Hospital, Detroit, MI.
 SO    Abstr Gen Meet Am Soc Microbiol. 1994;94:483 (abstract no. T-4). Unique
       Identifier : AIDSLINE ASM94/94313087
 AB    Measurement of the relative amounts of HIV unintegrated DNA (uDNA) and
       integrated DNA (iDNA) is a promising marker of therapeutic efficacy. The
       Hirt procedure for isolating total uDNA requires multiple extractions
       leading to DNA loss. We developed a novel column-based method for the
       isolation of cytoplasmic and nuclear uDNA fractions and the proviral
       iDNA from HIV-1. Aliquots of counted peripheral blood mononuclear cells
       (PBMCs) from 10 patients on antiretrovirals with CD4 counts > 200 were
       processed using the column or Hirt methods. The column method
       resuspended PBMCs in lysis buffer with the cytoplasmic fraction purified
       by centrifugation and ion exchange chromatography. The nuclear uDNA
       fraction was purified by base and detergent lysis followed by
       centrifugation and chromatography. The Hirt procedure lysed PBMCs with
       SDS and iDNA was separated from uDNA by high salt and centrifugation.
       The two fractions were digested and extracted multiple times. The amount
       of iDNA from both methods was quantified by measuring the OD260. The
       amount of HIV DNA in the uDNA and iDNA fractions was determined using
       quantitative PCR. The uDNA fractions were tested for iDNA contamination
       by RAS specific PCR. The nuclear uDNA fraction was tested for
       cytoplasmic uDNA contamination using mitochondrial DNA specific PCR. All
       10 of the patients had uDNA in both fractions. Seven of the patients had
       more uDNA in the nuclear fraction than the cytoplasmic fraction, 2 had
       about the same amount and 1 had more in the cytoplasm. There was an
       average of 10% uDNA in the cytoplasm and 26% in the nucleus. Two of the
       patients had recently switched antiretroviral therapy and their total
       uDNA levels dropped from 45% to 16% and 17% to 7%, respectively, after 4
       weeks on new therapy. The new method was found to recover approximately
       50 X more u/iDNA than the Hirt procedure. This new method has proven to
       be rapid, reliable and directly PCR compatible. We are using it to
       characterize the uDNA in the separated fractions and to monitor copy
       number changes in patients as therapy starts or changes.
 DE    Acquired Immunodeficiency Syndrome/*DRUG THERAPY/IMMUNOLOGY/
       MICROBIOLOGY  Cell Nucleus/MICROBIOLOGY  Cytoplasm/MICROBIOLOGY  DNA,
       Viral/ANALYSIS/*BLOOD/GENETICS  Human  HIV-1/GENETICS/*ISOLATION & PURIF
       Lymphocytes/IMMUNOLOGY/*MICROBIOLOGY  Polymerase Chain Reaction/*METHODS
       Sensitivity and Specificity  T4 Lymphocytes/IMMUNOLOGY  *Virus
       Integration  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
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