The complete human genome was too vast to be sequenced in a single pass during the Human Genome Project, so researchers had to discover a technique to divide it up. To accomplish this, they constructed a BAC library, a collection of DNA fragments.
BAC stands for Bacterial Artificial Chromosome.
BAC clones typically include DNA inserts up to 200,000 base pairs in length.
The bacteria are then cultivated to generate colonies in which each cell contains the same DNA fragment.
This is a BAC library, where BAC clones can be stored until they are required.
These segments are significantly easier to sequence than the full genome.
To create a genomic Bacterial Artificial Chromosome (BAC) library, it is necessary to first extract the cells having the desired DNA. Normal DNA sources for animal and human BAC libraries are white blood cells.
Then, these isolated cells are combined with hot agarose, a gelatinous substance.
The entire mixture is poured into a mould to create a series of little blocks containing thousands of the isolated cells each.
Enzymes are subsequently used to breakdown the cell membranes and release the DNA into the agarose gel.
Using a DNA-cutting enzyme, the DNA is sliced into pieces of around 200,000 base pairs in length.
These DNA-containing chunks of gel are then placed into pores in an agarose gel slab. Using electrophoresis, the DNA fragments are then sorted according to size.
On the opposite side of the gel, a'marker' solution is introduced. These are DNA fragments of a known size that can be utilised to identify DNA fragments of a specific size. This guarantees the BAC library is comprised of DNA fragments within a specific size range. These gel sections are excised, and the DNA fragments are retrieved.
Using the enzyme ligase, these DNA fragments are put into a BAC vector to unite the two pieces of DNA. Today, they are known as BAC clones.
BAC clones are introduced into bacterial cells, typically E. coli.
The bacteria are then distributed on nutrient-rich plates that only permit the growth of bacteria carrying BAC clones.
Each bacterial cell contains a copy of the BAC clone due to the rapid growth of the bacterium.
The bacteria are "selected" onto plates of 96 or 384 such that each tube carries a single BAC clone after their growth.
The germs can also be duplicated or frozen until researchers are ready to sequence the DNA.
The BAC library has been established.