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Rapid differentiation of bacterial species with multiple probes of different lengths in a single slot blot hybridization.

ABSTRACT: We describe a highly efficient method for dot and slot blot hybridizations with multiple oligonucleotide probes for high throughput identification of organisms and studies of microbial community structures. Several probes with distinct specificities were designed to have the same melting temperature but unique lengths by adding different numbers of nonspecific nucleotides to one end. All of the probes were mixed, labelled with 32P, and hybridized to one piece of membrane on which genes coding for 16S rRNAs from different bacterial species had been immobilized. After hybridization, the bound probes were eluted and resolved on a denaturing polyacrylamide gel and the identities of the genes coding for 16S rRNAs were read from an autoradiograph of the gel. The results from the application of this technique to pure actinomycete cultures are reported here.

SUBMITTER: Wang GC

PROVIDER: S-EPMC167738 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Rapid differentiation of bacterial species with multiple probes of different lengths in a single slot blot hybridization.

Wang G C GC Wang Y Y

Applied and environmental microbiology 19951201 12

We describe a highly efficient method for dot and slot blot hybridizations with multiple oligonucleotide probes for high throughput identification of organisms and studies of microbial community structures. Several probes with distinct specificities were designed to have the same melting temperature but unique lengths by adding different numbers of nonspecific nucleotides to one end. All of the probes were mixed, labelled with 32P, and hybridized to one piece of membrane on which genes coding fo ...[more]

PMID: 8534094

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Project description:BackgroundSothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments.The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample.Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing.ResultsWe have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time.We went on to experimentally validate a number of these automated designs by Southern blotting. The majority of probes we tested performed well confirming our in silico prediction methodology and the general usefulness of the software for automated genomic Southern probe design.ConclusionsSoftware and supplementary information are freely available at: http://www.genes2cognition.org/software/southern_blot.

Dataset Information

Rapid differentiation of bacterial species with multiple probes of different lengths in a single slot blot hybridization.

Publications

Rapid differentiation of bacterial species with multiple probes of different lengths in a single slot blot hybridization.

Similar Datasets

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