Colony hybridization background

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. 

A survey on molecular biology products (Ausubel et at, 1991) indicated that for nitrocellulose about 70% of respondents preferred S S NC (other manufacturers < 10%) but that the preference for nylon membranes differed little among Schleicher Schuell, Dupont NEN and Amersham. In the same survey, respondents had no clear preference for either nylon or nitrocellulose for Southern hybridization but a clear preference for nitrocellulose in the case of colony/plaque hybridization (where concentration of target is sufficient highly but background should be suppressed). 

Despite the wide use of radioprobes in colony or plaque hybridization assays, nonradioactive probes can be advantageous. The use of biotinylated probes, initially the most common among nonradioactive detection systems, is limited since biotin-streptavidin systems tend to give high background levels with bacterial material unless specific measures are taken. The more recently developed DIG (Table 7.2), but also other hapten-antibody systems such as sulfonated probes, are very attractive alternatives. The main restriction is that monoclonal antibodies (commercially available) should be used since polyclonal antisera often contain antibodies against bacteria. The main drawback of nonradioactive probes is the ability to reprobe the same membrane. It is possible, however, to strip a membrane of its probe after a colorimetric detection and to perform a chemiluminescent detection or vice versa. 

Hybridize for 2-3 h at 50°C. Hybridizing for more than 3 h will result in high background. Wash and expose to X-ray film to identify positive colonies. 

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