Introduction
Colony Blot
Hybridization is an application of nucleic acid
hybridization.
It is
combined with conventional environmental microbiological sampling.
It’s also used in viable plating procedures.
This procedure was developed by Grunstein and
Hogness in 1975.
Principle
The method
can be used to isolate any gene whose base sequence is represented in an
available RNA.
The
resulting DNA-prints of the colonies are then hybridized to a radioactive RNA
that defines the sequence or gene of interes.
The result of this hybridization is assayed by
autoradiography
Procedure
The
microbial colonies are transferred (blotted) to a membrane.
The cells
are lysed in place to release the nucleic acids
The RNA or
DNA (after denaturation) is fixed to the filter and hybridized with a labelled
probe.
Blocking
reagent may be added prior to the probe to prevent unspecific binding.
Excess probe
is washed away and the membrane is visualized by UV or autoradiography.
Colony blot
hybridization can be used for screening clones or bacterial isolates.
Application
Colony blot
hybridization techniques was investigated for potential in determining
populations of specific gene sequences in environmental samples.
Colony blot
hybridization has been used with environmental sample to enumerate
mercury-resistant bacteria in contaminated environments.
Various microorganisms with specific metabolic
activities of ecological importance have been detected using Colony blot hybridization. Example;
Pseudomonas fluorescens
Advantage
It
facilitates containment of any potentially hazardous hybrid plasmids that may
be cloned in such large screening operations.
By confining the reproductive state of the
hybrid-clones to colonies, the probability of escape is reduced over that for
liquid cultures because the number of bacteria per clone is generally smaller
and aerosols or accidental spills are less likely.
The
screening operation can be confined to small, controllable areas.
Discussion
Colony
hybridization of cloned hybrid plasmids can be used to isolate any gene, or
other DNA segment, whose base sequence is represented in an available RNA.
Colony blot
hybridization method using a fluorescently labeled oligonucleotide probe
specific for V. cholerae and V. mimicus.
This method is preferred to traditional
enrichment/selective medium isolation methods because the necessity for
extensive and laborious biochemical tests can be avoided.
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