What are BAC libraries?

What is a BAC?

• BAC stands for Bacterial Artificial Chromosome.
• These are small pieces of bacterial DNA that can be identified and copied within a bacterial cell.
• A BAC acts as a vector to artificially carry DNA into the cell of a bacterium, such as Escherichia coli, to make a BAC clone.

Plate growing Escherichia coli colonies
Image credit: Wikimedia Commons

• In general BAC clones carry inserts of DNA up to 200,000 base pairs long.
• The bacteria are then grown to produce colonies that contain the same fragment of DNA in each cell.
• The BAC clones can be stored until needed, this is a BAC library.
• These fragments are much simpler to sequence than trying to tackle an entire genome.

Making a BAC library

1. To make a genomic Bacterial Artificial Chromosome (BAC) library you first have to isolate the cells containing the DNA you want to store. For animal and human BAC libraries the DNA normally comes from white blood cells.
2. These isolated cells are then mixed with hot agarose, a jelly-like substance.
3. The whole mixture is poured into a mould to produce a set of small blocks, each containing thousands of the isolated cells.
4. The cells are then treated with enzymes to dissolve their cell membranes and release the DNA into the agarose gel.
5. A DNA-cutting enzyme is used to chop the DNA into pieces around 200,000 base pairs in length.
6. 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.
7. 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. This ensures that the BAC library is made from DNA fragments of a particular size range. These sections of the gel are cut out and the DNA fragments are extracted.
8. 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.
9. The BAC clones are added to bacterial cells, usually E. coli.
10. The bacteria are then spread on nutrient rich plates that allow only the bacteria that carry BAC clones to grow.
11. The bacteria grow rapidly, resulting in lots of bacterial cells, each containing a copy of the BAC clone.
12. After they have grown, the bacteria are then ‘picked’ into plates of 96 or 384 so that each tube contains a single BAC clone.
13. The bacteria can also be copied or frozen and kept until researchers are ready to use the DNA for sequencing.
14. A BAC library has been created.