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Gene Cloning

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Gene Libraries - Library Construction

The step following DNA extraction of an organism is the construction of a library to organize that DNA. A gene library can be defined as a collection of living bacteria colonies that have been transformed with different pieces of DNA from the organism that is the source of the gene of interest. If a library is to have a colony of bacteria for every gene, it will consist of tens of thousands of colonies or clones.

Library Construction


Restriction enzymes cut the extracted DNA sequences into gene-size pieces.
Constructing a gene library requires not only the extracted DNA, but also restriction enzymes and a plasmid. A gene library is made by cutting the extracted DNA into gene size pieces using restriction enzymes. These enzymes read the nucleotide sequence of the DNA, recognize specific sequences and then cut the DNA sequence by breaking the bonds between nucleotides in a DNA strand. The small DNA pieces are mixed into a test tube containing bacteria plasmids that have been cut with the same restriction enzyme.

Bacterial plasmids are cut with the same restriction enzymes. They are then combined with the extracted DNA and the two often anneal together.
A plasmid is an example of a vector, a mechanism for storing and transporting cloned DNA segments. Plasmids are small circles of DNA in bacterial cells that are naturally present in addition to the bacteria’s other DNA. They contain only a few of the thousands of genes that are present in a bacterium genome and can be copied many times in a single bacterial cell. In the library construction some of the enzyme cut DNA pieces will combine with the cut plasmids and form recombinant DNA (or DNA in a ’new combination’).

The recombinant plasmids are then transferred into bacteria using either electroporation or heat shock. Electroporation uses mild pulses of electricity to disrupt the cell walls of the bacterium and create small holes. The plasmids are small enough to pass through the holes into the cell. Heat shock works in a similar fashion. However, rather than using electricity to create holes in the bacterium, it is done by alternating the temperature between hot and cold.


After undergoing electroporation or heat shock, the bacteria are plated out onto petri dishes.
After undergoing electroporation or heat shock, the bacteria are plated out onto petri dishes. If each bacteria contains a different gene-size segment of the extracted DNA, it may take thousands of bacteria, and thus hundreds of plates, to contain all of the segments.

When the recombinant plasmid is inside the bacterium, the bacterium is fooled into thinking the new DNA is its own and begins replicating it. The plasmid can be replicated independently of the bacterium’s own DNA. The bacteria also multiply into colonies, with each colony coming from a cell that has been transformed with a different piece of recombinant DNA. The total of all of these colonies makes up the gene library. The scientist must then locate the colony that contains the gene of interest.



 

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