A simple method of detecting amplified DNA with immobilized probes on microtiter wells.
We have developed a simple hybridization method for the detection of specific DNA sequences amplified by polymerase chain reaction (PCR). This method is similar to an enzyme-linked immunosorbent assay (ELISA) format in that labeled PCR products at the 5' termini are hybridized with probes immobilized on a microtiter well and the bound PCR products are detected in a manner similar to that of an enzyme immunoassay (EIA). Two improvements have been made in immobilizing the probe to the microtiter wells, in terms of increasing both immobility and hybridization efficiency. One is that single-stranded (ss) DNA, without the complementary strand, is used. The other is that instead of a single copy, a tandem array of the probe is used for immobilization and hybridization. Using of ssDNA containing about a 60-repeat array of a relevant sequence as an immobilized probe, the sensitivity increased 10-fold over that of a single oligonucleotide unit. We also found that the hybridization conditions such as time, temperature, and solution composition could be simplified. Therefore this method is especially suited for handling of a large number of samples, for example detection of viruses, bacteria, and other pathogens, as well as mot human genetic disorders.
Kawai
S
S
Institute for Biotechnology Research, Wakunaga Pharmaceutical Co., Ltd., Hiroshima, Japan.
Maekawajiri
S
S
Yamane
A
A
D001483:Base Sequence; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral / Q000032:analysis*; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral