"Checkerboard" DNA-DNA hybridization.

TLDR

A method is introduced to hybridize many DNA samples with many probes on a single support membrane. The technique uses a Miniblotter 45 to fix up to 43 DNA samples in separate lanes, rotates the membrane to hybridize with 43 probes simultaneously, and employs a MiniSlot device for parallel lysate loading and horizontal lane deposition, with probes labeled digoxigenin or 16S rRNA oligonucleotides conjugated to alkaline phosphatase. The method enables simultaneous detection of multiple bacterial species in dental plaque, indicating potential for broader clinical and environmental applications.

Abstract

A method is introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single support membrane. Denatured DNA from up to 43 samples was fixed in separate lanes on a single membrane mounted in a Miniblotter 45. The membrane was then rotated 90 degrees in the same device, which enabled simultaneous hybridization with 43 different DNA probes. Hybridizations were also performed on lysates of bacterial cells blotted to membranes. A MiniSlot device allowed lysates loaded in parallel channels to be aspirated through the membrane, depositing horizontal lanes on the membrane surface. Hybridizations were performed in vertical lanes with either digoxigenin-labeled whole genomic probes or 16S rRNA-based oligonucleotide probes directly conjugated to alkaline phosphatase. The method permits the simultaneous determination of the presence of multiple bacterial species in single or multiple dental plaque samples, thus suggesting its usefulness for a range of clinical or environmental samples.