       Document 0327
 DOCN  M9640327
 TI    A comparative study of PCR product detection and quantitation by
       electro-chemiluminescence and fluorescence.
 DT    9604
 AU    Yu H; Bruno JG; Cheng TC; Calomiris JJ; Goode MT; Gatto-Menking DL;
       Optech Corporation, San Antonio, TX 78229, USA.
 SO    J Biolumin Chemilumin. 1995 Jul-Aug;10(4):239-45. Unique Identifier :
       AIDSLINE MED/96084083
 AB    Amplification and detection of target DNA sequences are made possible in
       a polymerase chain reaction (PCR) by using a mixture of biotinylated and
       ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers. In this
       way, biotin for capture and Ru(bpy)3(2+) for detection are directly
       incorporated into the PCR product obviating subsequent probe
       hybridization. PCR of a bacterial DNA template from Alteromonas species
       strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled
       primers amplified a 1 kilobase region. Serial dilution of PCR product
       followed by magnetic separation with Streptavidin (SA)-coated magnetic
       beads and an electrochemiluminescence (ECL) assay using the
       semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA
       detection. ECL assay of probe hybridization to a human immunodeficiency
       virus (HIV) sequence also produced pg level sensitivity. Quantitative
       DNA determination by ECL assay correlated well with visual detection of
       DNA in electrophoretic gels. However, DNA detection by ECL assay was 10
       to 100 times more sensitive than conventional ethidium bromide staining.
       The combination of DNA-based magnetic separation with ECL assay provides
       a very sensitive and rapid method of quantitating DNA which, owing to
       its rapid and facile nature, may have many applications in the research,
       environmental monitoring, industrial and clinical fields.
 DE    Base Sequence  *Chemiluminescence  Comparative Study  DNA
       Primers/CHEMISTRY/GENETICS  DNA, Bacterial/ANALYSIS/GENETICS  DNA,
       Viral/GENETICS  Ethidium  Fluorescence  Fluorescent Dyes  Genes, gag
       Gram-Negative Aerobic Bacteria/GENETICS  HIV/GENETICS  Molecular
       Sequence Data  Polymerase Chain Reaction/*METHODS/STATISTICS & NUMER
       DATA  Sensitivity and Specificity  JOURNAL ARTICLE

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

