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The BACs, with their inserted DNA, are then taken up by bacterial cells. A big amount of DNA may be modified such that it can be transmitted in bacteria as a circular artificial chromosome known as a bacterial artificial chromosome, or BAC. BACs can include segments of an organism’s DNA ranging in size from 100,000 to 300,000 base pairs. White blood cells provide the DNA for both animal and human BAC libraries. These DNA pieces are joined together using an enzyme called ligase before being introduced into a BAC vector. They’re now known as BAC clones.
Bacterial Artificial Chromosome
What is Bacterial Artificial Chromosome?
Each BAC is a cloned DNA clone with between 100 and 300 thousand base pairs of cloned DNA. Because the BAC is considerably smaller than the natural bacterial chromosome, it is simple to separate the BAC DNA from the rest of the bacterial cell’s DNA and get pure cloned DNA. BACs are particularly useful for mapping and sequencing mammalian genomes because of this and other significant properties.
BACs (bacterial artificial chromosomes) are huge genomic clones that are kept stable in E. coli. BACs can be used to get gene-specific promoter sequences since they are typically big enough to include a whole gene and its accompanying regulatory components. Due to the huge size of BACs, recombination-based approaches to change these DNAs have been developed. Transgenic mice with reporter gene-engineered BACs have been proven to label distinct populations of cells. As a result, BACs serve as a tool that should make manipulating gene expression in specific cell types in the nervous system much easier.
How are Bacterial Artificial Chromosomes created ?
To create a genomic Bacterial Artificial Chromosome (BAC) library, separate the cells that contain the DNA you wish to save. White blood cells provide the DNA for both animal and human BAC libraries. These separated cells are then combined with a jelly-like material called hot agarose.
The entire mixture is poured into a mould, resulting in a series of little blocks containing thousands of isolated cells apiece. After that, enzymes are used to disintegrate the cells’ cell membranes and release the DNA into the agarose gel. An enzyme that cuts DNA is used to cut the DNA into fragments that are approximately 200,000 base pairs long.
These gel blocks with diced up DNA are then placed into pores in an agarose gel slab. A solution of markers is introduced on the other side of the gel. These are known-size DNA fragments that may be used to help identify DNA fragments of a specific size. This guarantees that the BAC library is composed of DNA fragments in a specific size range. The DNA fragments are retrieved from these areas of the gel.
These DNA pieces are joined together using an enzyme called ligase before being introduced into a BAC vector. They’re now known as BAC clones. The BAC clones are frequently introduced to E. coli bacterium. The bacteria are then distributed on nutrient-rich plates, where only bacteria carrying BAC clones may proliferate.
The bacteria multiply quickly, resulting in a large number of bacterial cells, each with a copy of the BAC clone. The bacteria can also be duplicated or frozen and stored until the researchers are ready to sequence the DNA. There is now a BAC library.
Note on DNA and BAC
When compared to viral and nonviral cDNA vectors, gene expression from bacterial artificial chromosome (BAC) clones has been shown to facilitate physiologically appropriate levels. BACs are big enough to transmit entire genes in their natural chromosomal position, along with adjacent regulatory elements, to give all of the signals necessary for proper spatiotemporal gene expression. Until recently, the use of BACs for functional investigations was limited due to their huge size, which made alterations using traditional genetic engineering procedures difficult.
The introduction of in vivo homologous recombination technologies based on E. coli recombination has aided in the resolution of this challenge by allowing for the simple manufacture of high molecular weight BAC DNA without the need of restriction enzymes or cloning processes.
Recombineering in BAC modifications has a wide range of applications. Depending on the design and nature of the targeting substrate and target site, recombineering can be used in a variety of mutagenesis techniques; read the text for additional information. Stippled boxes indicate homologous recombination sequences. Gene substitution.
Any sequence of interest can be used to replace a target site via recombineering. Insertion. Recombineering can also be used to add DNA without deleting any of the current sequence.
Selection/reverse selection Recombineering can mediate small alterations like nucleotide substitutions by introducing a selective cassette and then replacing it with the changed version of the target site in two rounds of recombinations.
Conclusion
BAC is considerably smaller than the natural bacterial chromosome, it is simple to separate the BAC DNA from the rest of the bacterial cell’s DNA and get pure cloned DNA. BACs are particularly useful for mapping and sequencing mammalian genomes because of this and other significant properties.
