Highly sensitive detectiong of DNA using enzyme-liked DNA-probe. I Colorimatric and fluorometric detection

Abstract

In order to develop non-radioactive oligonucleotide derivatives and to examine their utility as a diagnostic tool, namely as DNA-probe, an enzyme-linked oligonucleotide was synthesized. Oligonucleotide complementary to M13mp8 phage DNA was linked to alkaline phosphatase via a crosslinker and a spacer. M13mp8 phage DNA (single strand) immobilized on the nitrocellulose membrane was hybridized with the enzyme-linked oligonucleotide. The hybrid was detected with three detection methods; (1)colorimetric detection in solution, (2)colorimetric one on membranes, and (3)fluorometric one in solution. Methods(2) and (3) gave high sensitivities to detect as low as several to several tens attomoles of DNA and it was found that those methods with enzyme-linked oligonucleotides are potent for DNA-probe methodology from the viewpoint of automation.