Structural Biochemistry/DNA recombinant techniques/Detecting specific genes

Overview

A genomic library is a collection of all the genes of an organism. After creation of a genomic library, a particular gene usually needs to screened from the genomic library just created. A method to do this is to carry out a colony hybridization experiment.

Colony Hybridization Experiment

This technique is used to screen for a plasmid based genomic library. Bacteriophage plaques are screened. First, bacteria is transformed with a plasmid by using a bacteriophage to infect the colony. These are plated on a petri dish and incubated so that phage plaques are allowed to form. A plague with an identical phage will develop on the petri dish wherever the phage is located and infected by the bacterium. Next, replication of the original plate is made by placing a nitrocellulose sheet on top of the original plate. This allows the plaques to be transferred in a certain array. Both infected bacteria and phage DNA will be release from the lysed cells to attach to the nitrocellulose sheet to form spots. This is then treated with 2M NaOH to lyse the phages and separate the strands of DNA. When the DNA is denatured by the 2M NaOH treatment, the DNA strand is more accessible to be hybridize with the 32P probe. One must next neutralize the dish and then allowed to dry to immobilize the DNA. This is then mixed with a 32P probe and allowed to anneal to target any DNA sequence present on the nitrocellulose sheet. This is then subject to autoradiography by placing it on an x-ray film. The spots show where the probe has hybridized with the DNA and this is used to locate the genomic clone from the plaques on the original petri dish.