Rapid electrochemiluminescence assays of polymerase chain reaction products.
We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.
Kenten
J H
JH
Department of Molecular Biology, IGEN Inc., Rockville, MD 20852.
Casadei
J
J
Link
J
J
Lupold
S
S
Willey
J
J
Powell
M
M
Rees
A
A
Massey
R
R
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D001483:Base Sequence; D056831:Coordination Complexes; D015342:DNA Probes; D004563:Electrochemistry; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis; D027383:Papillomaviridae / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017975:Ruthenium Compounds
D015082:2,2'-Dipyridyl; D056831:Coordination Complexes; D015342:DNA Probes; D009841:Oligonucleotides; C547232:tris(2,2-bipyridine)-ruthenium(II); D017975:Ruthenium Compounds