Biotechnology

BAC Clones

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A bacterial artificial chromosome (BAC) is an artificially engineered DNA molecule that is used to clone DNA sequences in bacterial cells. BACs are often used in connection with DNA sequencing. Parts of an organism’s DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs. The BACs, with their inserted DNA, are then taken up by bacterial cells. As the bacterial cells grow and divide, they amplify the BAC DNA, which can then be isolated and used in sequencing DNA.

A large piece of DNA can be engineered in a fashion that allows it to be cultivated as a circular artificial chromosome in bacteria so called bacterial artificial chromosome, or BAC. Because the BAC is much smaller than the endogenous bacterial chromosome, it is straightforward to purify the BAC DNA away from the rest of the bacterial cell’s DNA, and thus have the cloned DNA in a purified form. This and other powerful features of BACs have made them much useful for mapping and sequencing mammalian genomes.

Formation a BAC library

·   First we have to separate the cells having the DNA we want to store.

·   These isolated cells are then mixed with hot agarose, a jelly-like substance.

·   The whole mixture is poured into a mould to produce a set of small blocks, each containing thousands of the isolated cells.

·   The cells are then treated with enzymes to dissolve their cell membranes and release the DNA into the agarose gel.

·   A DNA-cutting enzyme is used to chop the DNA into pieces around 200,000 base pairs in length.

·   These blocks of gel containing chopped up DNA are then inserted into holes in a slab of agarose gel. The DNA fragments are then separated according to size by electrophoresis.

·   On the other side of the gel a solution of ‘markers’ are inserted. These are DNA fragments of known size which can be used to help identify fragments of DNA of a particular size.

·   These DNA fragments are inserted into a BAC vector using an enzyme called ligase to join the two bits of DNA together. They are now called BAC clones.

·   The BAC clones are added to bacterial cells, usually E. coli.

·   The bacteria are then spread on nutrient rich plates that allow only the bacteria that carry BAC clones to grow.

·   The bacteria grow rapidly, resulting in lots of bacterial cells, each containing a copy of the BAC clone.

·   After they have grown, the bacteria are then ‘picked’ into plates of 96 or 384 so that each tube contains a single BAC clone.

·   The bacteria can also be copied or frozen and kept until researchers are ready to use the DNA for sequencing.

·   A BAC library has been created.

Identification of bacterial artificial chromosome (BAC) clones containing specific sequences is a prerequisite for many applications, such as physical map anchoring or gene cloning. Existing BAC library screening strategies are either low-throughput or require a considerable initial input of resources for platform establishment. 

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