Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains

TLDR

Fluorometric hybridization in microdilution wells was developed to determine genetic relatedness among microorganisms. The method labels total chromosomal DNA with photoreactive biotin, hybridizes it to immobilized single‑stranded DNA in microdilution wells, and quantitatively detects bound biotinylated DNA with β‑galactosidase and a fluorogenic substrate, comparing homology values to membrane‑filter assays. The results indicate that this fluorometric direct‑binding approach can serve as an alternative to radioisotope‑based and membrane‑filter hybridization methods.

Abstract

Fluorometric hybridization in microdilution wells was developed to determine genetic relatedness among microorganisms. Total chromosomal deoxyribonucleic acid (DNA) for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with single-stranded unlabeled DNAs which had been immobilized on the surfaces of microdilution wells. After hybridization, biotinylated DNA was quantitatively detected with beta-D-galactosidase and a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactopyranoside. Homology values obtained with this fluorometric direct binding method were compared with values obtained with two membrane filter methods, one in which photobiotin labeling was used and one in which radioisotope labeling was used. The results showed that the fluorometric direct binding method in which microdilution wells are used could be an alternative to radioisotope and membrane filter hybridization methods.