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.Quantitative measurement of deoxyribonucleic acid (DNA)-DNA hybridization from renaturation rates has contributed to determinations of genetic relatedness among bacterial strains (2,4,5, 13,19). The methods usually used in this technique are either a free solution method in which S1 nuclease, (4), spectrophotometry, (5, 19), or hydroxyapatite is used (2) or a method in which single-stranded DNA is fixed on a solid support, such as nitrocellulose filters (1, 12). However, to carry out most of these hybridization experiments, DNA must be labeled with radioactive substances by nick translation (18) or random primed labeling (9). Recent developments have made it possible to label DNA with nonradioactive materials without using enzymes (10,11,17). Biotinylation of DNA with photoreactive biotin (photobiotin) (10) is one of these recent developments. In this procedure DNA is labeled by mixing it with photobiotin and irradiating the mixture with sunlight for 15 min. Biotinylated DNA has been used for Southern and dot blot hybridizations (10,14). Hybridized DNA fragments are usually visualized with alkaline phosphatase or peroxidase color detection methods (10,14,17). We used photobiotinylated DNA for quantitative detection of DNA-DNA hybridization to determine genetic relatedness among microorganism for taxonomic studies (4, 5,19). Quantitative hybridization was carried out in microdilution wells. After hybridization, streptavidin-conjugated beta-D-galactosidase was bound to photobiotinylated DNA. A sensitive fluorogenic substance, 4-methylumbelliferyl-beta-~-galactopyranoside was used as a substrate (16). In this paper the fluorometric hybridization method is compared with a radioisotope method (19) as an alternative procedure to determine genetic relatedness among bacteria. (Table 1) were prepared by the procedures of Marmur, with minor modifications (8). Under optimal conditions, 100-pl portions of a heat-denatured, purified reference DNA solution (20 pg of DNA per ml) in * Corresponding author.
MATERIALS AND METHODS
DNAs of bacterial strainsphosphate-buffered saline (...
