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Saturday, 19 January 2019

COLONY BLOT HYBRIDIZATION

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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