Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products, using 3'-labelled oligonucleotide probes.
Described are rapid assays for the analysis of PCR products in a one step, non-separation assay based on the use of electrochemiluminescence generated from a tris-bipyridine ruthenium (II) label. The assay uses PCR incorporation of a biotinylated oligonucleotide as a primer, with the inclusion of a labelled oligonucleotide. Oligonucleotides were labelled with an N-hydroxy succinimide ester of tris-bipyridine ruthenium (II) dihexafluorophosphate (Origen-label) by modifying the 3' and 3' 5' ends of the oligonucleotide probes. The assay makes use of the inherent thermal stability and absence of polymerase activity on such probes to allow the PCR and probe hybridization to be completed automatically on the thermocycler. The assay is concluded by the addition of PCR samples to streptavidin beads on an electrochemiluminescence analyser for binding and analysis. Target genes evaluated were the HIV-1 gag gene, and cystic fibrosis delta F-508 deletion mutation. The results obtained from these assays demonstrated the detection of 10 copies of the HIV-1 gag gene, and cystic fibrosis delta F-508 mutation in 1 ng of human DNA within 15 min. This assay format allows a rapid and simple determination of specific amplified DNA sequences, reducing the contamination risks due to washes and multiple pipetting.
Gudibande
S R
SR
Department of Molecular Biology, IGEN Inc., Rockville, MD 20852.
Kenten
J H
JH
Link
J
J
Friedman
K
K
Massey
R J
RJ
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D001710:Biotin; D001798:Blood Proteins / Q000235:genetics*; D040501:Calgranulin A; D003550:Cystic Fibrosis / Q000453:epidemiology / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes; D005813:Genes, Synthetic; D015750:Genes, gag; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D015994:Incidence; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D009942:Organometallic Compounds; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001798:Blood Proteins; D040501:Calgranulin A; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009942:Organometallic Compounds; C026422:cystic fibrosis serum factor; C079178:Origen (label); D015082:2,2'-Dipyridyl; D001710:Biotin; D004247:DNA