Colony blot hybridization

Colony blot hybridization for detection of cells carrying a plasmid clone of the p-chain gene of hemoglobin. 

Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-... 

Single cell-derived colonies or foci are readily analyzed by cell colony hybridization (Avraham et al., 1989). Although cell colony hybridization on animal cells was described early (Villarreal and Berg, 1977), the subsequently developed dot/slot blot hybridization is normally preferred. Colony hybridization can have some particular advantages, however, such as the quantification of transfected cells or foci, the isolation of certain foci or colonies and may be useful for screening of stable somatic cell hybrids. 

Following transformation of mutant S. 6803 T-1,2,3 [4] with plasmid pSF8.1 colonies were selected for photoautotrophic growth. DNA from potoautotrophic transformants were then analyzed by Southern blot hybridization to confirm the insertion of the A. hybridus psbA gene. Fig. 2 shows the results from one selected transformant, S. 6803 SF. 

A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper, which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. 

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... 

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. 

Bacterial cells are plated onto a solid medium that only allows the growth of transformed cells. Once the colonies become visible, the plate is blotted with a nitrocellulose filter. The cells clinging to the filter are lysed and the released DNA is denatured and deproteinized. After a labeled probe is added, unhybridized probe molecules are washed away. Cells that possess DNA sequences that hybridize with the probe are identified by comparing the autoradiogram of the filter with the master plate. 

Probes labeled directly with HRP have been used in many membrane hybridization applications (5,6), including Southern blots. Northern blots, colony and plaque screening, PCR product detection/ identification, YAC clone screening, and RFLP analysis. 

The use of probes directly labeled with horseradish peroxidase (HRP) in conjunction with enhanced chemiluminescence (ECL) allows a flexible approach to hybridizations and detections. This is shown by the diversity of applications in which this labeling and detection system has been used. The applications include Southern blots (1,2) and Northern blots (3), colony and plaque screening for positive clones (4), YAC clone screening (5), and PCR product detection (6,7). 

Clones containing genomic DNA of a particular sequence may be identified by plaque hybridization, which is similar in principle to colony hybridization for detection of particular plasmid recombinants. A lawn of E. coli containing several thousand plaques of independent recombinants is blotted on to a nitrocellulose filter. The DNA is released from the blotted recombinant phage, denatured by alkali, and neutralized. A radioactive probe of nucleic acid containing sequences of the gene of interest is then hybridized to the DNA plaques on the filter. Positive plaques, detected by autoradiography as radioactive DNA-RNA or DNA—DNA hybrids, are then picked off and amplified at lower plaque density (approx. 100 per plate). 

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... 

---