       Document 0552
 DOCN  M9610552
 TI    Use of a rapid and simple method to extract proviral DNA in the
       identification of HIV-1 by PCR.
 DT    9601
 AU    Tagliaferro L; Corbelli M; Maietta G; Pellegrino V; Pignatelli P; Dept.
       Virology, Laboratory Pignatelli, Lecce, Italy.
 SO    New Microbiol. 1995 Jul;18(3):303-6. Unique Identifier : AIDSLINE
       MED/96028328
 AB    DNA extraction is a critical step in PCR analysis and is closely related
       to its sensitivity. Traditional methods, based on phenol-chloroform
       extraction, require more time and the use of toxic reagents.
       GeneReleaser (Bio Ventures Inc.) is a commercial product which releases
       DNA from whole blood, cell cultures, bacterial colonies and the like.
       Cells lysis and DNA extraction are accomplished directly in the
       amplification tube on a thermocycler. We used GeneReleaser in the
       identification of HIV-1 proviral DNA by PCR on whole blood samples. All
       samples arrived at our laboratory for HIV-1 detection were treated with
       two different procedures. The classical one was based on the lysis of
       separated lymphocytes by proteinase K, while the other consisted in DNA
       extraction by GeneReleaser from 5 microliters of whole blood in sodium
       citrate. All samples were amplified for HIV-1 GAG region; to prevent
       carry-over contamination Uracil N-glycosylase (UNG) sterilization was
       performed. Amplified sequences were revealed using the DEIA commercial
       system (Sorin Biomedica, Italy). To verify the suitability both of cell
       lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a
       specific sequence of HLA-DQ-alpha gene. Initial data indicate that this
       new method might reduce the performance time of PCR (DNA extraction time
       was around 15 minutes) and improve PCR sensitivity.
 DE    Adult  Blood Cells/VIROLOGY  Comparative Study  DNA/ISOLATION & PURIF
       DNA Primers  DNA Repair  Female  Genes, gag  Human  HIV
       Infections/BLOOD/*DIAGNOSIS  HIV-1/*ISOLATION & PURIF  HLA-DQ
       Antigens/GENETICS  Infant, Newborn  Lymphocytes/VIROLOGY  Male
       Nucleosidases/CHEMISTRY  Polymerase Chain Reaction/*METHODS
       Proviruses/*ISOLATION & PURIF  Serine Proteinases/CHEMISTRY  JOURNAL
       ARTICLE

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

