Colony hybridization
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Colony hybridization is a method of selecting bacterial colonies with desired genes through a straightforward cloning and transfer process.[1] The genes of interest have been added to a bacterial plasmid previously through recombination, allowing genes from other organisms to be analyzed within a bacterial colony. The overall process involves a transfer of genetic material from one medium to another, typically using nitrocellulose filter paper, with the intended goal of identifying and isolating a specific gene. Radiographed RNA is used to find the desired sequence within the new bacterial colony and essentially "light it up" so that the sequence can be identified for transfer. The most common purpose of colony hybridization is to verify that a certain DNA sequence was able to successfully enter into a new cell, meaning that the cells being analyzed through this method are the result of recombination between a specific piece of DNA and a bacterial plasmid.[2] This method was discovered by Michael Grunstein and David S. Hogness. [3]
Colony hybridization begins with a desire to extract a segment of DNA containing a specific gene, such as a gene that conveys antibiotic resistance.[4] A specific piece of DNA is removed from its respective cell culture and inserted into a bacterial plasmid via a process known as recombination. These bacterial plasmids are cultured on a nutrient agar plate, leading to the formation of bacterial colonies, some of which ideally continue to contain the gene of interest. A nitrocellulose filter is then washed three times with distilled water, placed in between absorbent sheets, and heated at high temperatures to kill bacteria or other microorganism. This filter, which has a pore size of .45 μm, undergoes these processes to ensure that there is no contamination during the transfer, thus allowing for accuracy in results. The bacterial colonies are then symmetrically replicated onto the nitrocellulose filter by direct contact. At this point, the cells on the filter membrane are lysed in order to open up the plasmids for easier access and their DNA is denatured, which allows it to bind to the filter.[2] These DNA clusters are then hybridized to a desired radioactively-labelled RNA or DNA probe and screened by autoradiography. The RNA (or DNA) probe was selected specifically beforehand based on the DNA carrying the desired gene, since the probe must contain the complementary strand that will allow it to bind accurately to the correct genetic material. Through use of the radioactive probe, the clusters that exhibit the desired gene are identified to be used in further research. Frequently, they are then matched up to the corresponding (living) bacterial colonies, which have not undergone the cell lysis procedure, which may also be isolated for further growth and experimentation.[3]