Colony hybridization method

The original colony hybridization method described the use of radio-labeled probes and the detection of positive hybridization events by autoradiography (1). However, because of the high waste disposal costs, short half-lives, long autoradiographic exposures, and potential health hazards associated with radioisotopes, there is interest in alternative methods to detect positive hybridizations. 

Maas, R. (1983) An improved colony hybridization method with significantly increased sensitivity for detection of single genes Plasmid 10, 296-298. 

PCR is a useful tool, especially due to its great sensitivity and speed. This method complements the colony hybridization method by specific probes, and the two methods permit the early detection (PCR) and quantification (specific probes) of bacterial strains that alter wine. However, in the near future, the more recently developed quantitative PCR in real time is likely to provide quantitative data with all the accuracy and speed of PCR. In the future, other methods of genome analysis will probably permit the identification of species less common to wine with greater certainty. These species are interesting because of their metabolism or Iheir resistance to wine... 

Grunstein, M., and Hogness, D. S., 1975. Colony hybridization A specific method for the isolation of cloned DNAs tliat contain a specific gene. Proceedings of the National Academy of Sciences U.S.A. 72 3961—3965. Article describing die colony hybridization technique for specific gene isolation. 

DNA adsorption properties were first studied using a variety of solid supports for classical analysis methods including Southern and Northern transfers, dot-blotting, colony hybridization and plaque-lifts [31,32]. Studies of the interactions between nucleic acids and nitrocellulose revealed that molecular weight, finite macromolecular conformation, ionic forces and weaker forces of attraction all play a role. DNA is retained on nitrocellulose only in... 

C.A. Pettigrew and G.S. Sayler. 1986. Application of DNA colony hybridization to the rapid isolation of 4-chlorobiphenyl catabolic phenotypes. J. Microbiol. Methods. 5 205-213. 

Pettigrew, C. A. Sayler, G. S. (1986). The use of DNA DNA colony hybridization in the rapid isolation of 4-chlorobiphenyl degradative bacterial phenotypes. Journal of Microbiological Methods, 5, 205-13. 

To facilitate our work on plasmids with no known phenotype, we have developed a method for the use and detection of biotinylated probes in colony hybridization. It is suitable both for the detection of rare positive hybridization events over a background of nonreactive colonies and for the detection of nonhybridizing colonies in a population containing sequences homologous to the probe. The latter capability could be useful in such applications as the detection of cured (i.e., plasmid-free) cells in a bacterial population containing plasmids. 

Lonvaud-Funel et al. (1989, 1991a) described the identification of LAB during vinification and wine storage by DNA-DNA hybridization. Genomic DNA of the strain to identify was hybridized with total genomic DNA probes extracted from reference strains. They found that this method was particularly efficient when used in colony hybridization to study mixed populations at least five different species can be detected in a mixture with this system (Lonvaud-Funel et al. 1991b). 

Grunstein, M., Hogness, D. (1975) Colony Hybridization A. Method for the Isolation of Cloned DNAs that Contain a Specific Gene, Proc. Natl. Acad. Sci. USA 72,3961-3966. 

Make ds cDNA using random primers (Section 4.4) and clone according to standard methods (Section 4.5). For colony hybridization, use enriched (after step 5) and unenriched probes (after step 1) on parallel blots. 

In another approach, the colony hybridization technique (Figure 18D, bacteria are screened by using a radioactively labeled nucleic acid probe, an RNA molecule or a single-stranded DNA molecule with a sequence complementary to that of a specific sequence within the recombinant DNA. Bacterial cells are plated out onto solid media in petri dishes and allowed to grow into colonies. Each plate is then blotted with a nitrocellulose filter. (Most of the original colonies remain on the petri dishes.) The cells on the nitrocellulose filter are lysed, and the released DNA is treated so that hybridization with the probe can occur. Once nonhybridized probe molecules have been washed away, autoradiography (Biochemical Methods 2.1) is used to identify the colonies on the master plate that possess the recombinant DNA. 

Grundstein M, Hogness DS (1975) Colony hybridization a method for the isolation of doned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72 3961-3965... 

Any method may be used to generate the radioactive probes required at various stages of the protocol presented The Pnme-It II Kit available from Stratagene reliably generates high specific-activity probes useful for the hybridizations utilized m the colony hybridization and Northern blotting procedures... 

Colony hybridization is a relatively quick and inexpensive method for estimating the number of organisms of a given systematic group (when 16S gene is targeted) or canying... 

At present in enology, two particular cases are analyzed in this manner strains of Pediococcus damnosus, responsible for ropiness disease, and strains which produce histamine, notably 0. oeni. Preliminary studies have shown that P. damnosus strains capable of synthesizing the ropy wine polysaccharide possess a supplementary plasmid, contrary to normal strains. The ropy character is linked to the presence of this plasmid. A fragment was cloned in E. coli and now constitutes the base material for preparing the probe. In this manner, colony hybridization permits the identification of ropy clones even when mixed with other Pediococcus clones or other species of bacteria. This method is routinely used to identify this undesirable population in the microflora of wines at the end of winemaking and during aging (Lonvaud-Funel et al, 1993). 

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