Colony hybridization technique

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. 

Limitations notwithstanding, colony hybridization techniques have been used successfully for a number of years to monitor environmental samples. Representative examples include detection of naphthalene and toluene degraders in contaminated soils (Sayler et al., 1985), recombinant organisms in groundwater (Jain et al., 1987 ... 

The bacterial colony hybridization technique can be adapted easily to eukaryotic cells (Avraham et al., 1989). Nitrocellulose membranes, autoclaved for 30 min, are placed in a sterile plate containing sterile PBS and the monolayer cultures are washed with sterile PBS twice (PBS is Ca and Mg free). Three drops of PBS are added to each plate and the membrane is placed on top of the cells and left for 2 min (no air bubbles both membrane and plate marked). The membranes are then gently lifted and fresh medium is added to the cells to regenerate in a 37 C incubator. 

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. 

Probing of the genes for the purpose of organism detection and classification can be done in either the community DNA (i.e., following DNA extraction and amplification) or in the DNA of a specific organism, such as probing of the DNA in a colony hybridization technique. 

Screeiring plasmid-based genomic libraries often uses a technique termed "colony hybridization." This techirique can employ autoradiographic films, which detect ... 

DNA hybridization assays rely on the properties of single strand DNA to hybridize with complementary sequences. They also use the property of DNA to bind to nitrocellulose membranes (Bouvet and Vemozy-Rozand 2000). The availability of cloned stxl and stx2 genes allow the development of DNA probes for the detection of STEC. Initially, probes labeled with or were used to screen a large number of E. coli fecal isolates by colony hybridization. These techniques were specific and sensitive and could differentiate between stxl and stx2 if stringent conditions were used. 

Colony hybridization A technique that is used to screen bacteria for the presence of a specific recombinant DNA sequence. Colonies of the bacteria are transferred to a filter, treated to lyse the cells and denature the DNA, and then exposed to a labeled DNA probe that is complementary to part of the sequence in question. Colonies that bind the probe possess the sequence. 

Each sample was diluted in decimal stages to ensure the reliability of the end result. The dilute solutions were seeded evenly on the surface of the Petri dishes with sterile beads. This technique is necessary if the cells are to be identified by DNA/DNA hybridization (Volume 1, Section 4.3.5) on colonies or PCR (Volume 1, Section 4.3.6). If the wine contains very few microorganisms, it is filtered onto a 0.45-tim membrane, which is then deposited on the specific... 

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