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Uploaded byManimaran G
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Colony hybridisation

This document summarizes the process of colony hybridization. Colony hybridization allows researchers to select bacterial colonies containing specific genes. The procedure involves lysing bacterial colonies on a nitrocellulose filter, denaturing the DNA, and hybridizing the DNA to a labeled probe for the target gene. Unbound probe is then washed away. Where the probe binds to colonies containing the target gene, dark spots will appear on an x-ray film placed over the filter, allowing identification of recombinant colonies containing the desired gene. Colony hybridization has applications in identifying recombinant bacteria, cytogenetics studies, disease diagnosis, fingerprinting, and screening bacterial colonies.

Education◦
By G.MANIMARAN I st Msc Biotechnology
SYNOPSIS ▫ INTRODUCTION ▫ PRINCIPLE ▫ PROCEDURE ▫ APPLICATIONS 2
introduction ▫ Colony hybridization is a method of selecting bacterial colonies with desired genes. This method was discovered by Michael Grunstein and David S. Hogness in 1975 3
definition ▫ Colony hybridization can define as the method for the isolation of the specific DNA sequences or genes from the bacterial cells containing hybrid DNA, by the means of a nitrocellulose membrane filter. The transferring medium then goes through several chemical and physical treatments. 4
principle ▫ Selection of Recombinant bacteria by hybridization of probe DNA with DNAs of bacterial colonies on a nitrocellulose Filter is called colony hybridization ▫ This used to pick out recombinant bacteria has rDNA that has specific gene 5
procedure ▫ The transformed bacterial culture is diluted and poured on agar plate and incubated till colony appeared 6
Colonies form the master plate is transferred to replica plate marked the position A Nitrocellulose Filter is Placed over The Replica Plate 7
After the nitrocellulose paper taken from the petridish and placed in alkai solution (0.5n) NAOH The alkali solution lyses bacterials cells and denature DNA 8
Filter kepts dipped into proteinase k solution It removes protein from the filter dna bound alone filter 9
it was fixed with filter by drying at 80o c inbetween filter papers the step called baking . Consequently the filter has dna print of the bacterial colonies. The filter has dna print of bacterial colonies 10
then filter placed in hybridization solution containing dna probes or rna probes labelled with p32 isotopes forms dna-dna hybrid Then filter washed with saline citrate solution to remove unbound probes 11
an x-ray film is placed over the filter for about 4 hours to get an autoradiogram the probe makes dark spot on x-ray film 12
the autoradiogram is matched with the master plate the recombinant cells contain the particular desired gene. 13
the recombinant colonies are picked up and cultured for future use 14
application  identification recombinant bacterial colonies by hybridization of probe dna or rna  it used in cytogenetics studies  it is used for the diagonosis of infectious diseases.  it is used In finger printing  it is used as screening method of bacterial colonies 15
▫ Procedure diagram 16
Thank you 17