The Importance of BACs in Genome Sequencing (and Why They've Replaced YACs)

To successfully clone a gene, you’ll need a carrier DNA (vector) to take the DNA sequence into the target cell. Previously, plasmids and cosmids were used for this purpose, since plasmids can hold up to 10,000 nucleotide base pairs (or 10 kilobase pairs), while cosmids can hold up to up to 30 kb. In comparison, the average size of the human gene is about 27 kb, while some viruses have significantly larger genomes. For example, adenoviruses can be about 40 kb, while the pox virus and herpes virus can have a genome size of about 230 to 250 kb.

With these obvious limitations, biologists knew they needed something that can hold larger pieces of DNA so they can study several genes and maybe even the entire viral genome at once. This realization led to the creation of bacteria artificial chromosomes, or BACs. As BACs were specifically designed to hold larger pieces of DNA, they are often used in DNA sequencing projects (e.g., the Human Genome Project).

What are bacterial artificial chromosomes (BACs) and what role do they play in genome sequencing? To answer this question, let’s take a closer look at what BACs are.

Bacterial Artificial Chromosomes, Explained

Generally, BACs are artificially engineered DNA segments used primarily to alter and clone genes in bacteria (particularly E. coli). Since they are based on fertility plasmids (F-plasmid or F’) that allow the transfer of genes between cells, these DNA constructs ensure the even distribution of plasmids following cell division.

There are several reasons why the F-plasmid was used in creating BACs. Specifically, the F-plasmid:

• can hold up to 350 kb of DNA
• has replication origins and capable of regulating copy number
• has well-defined plasmid partitioning and chromosome segregating systems (parA and parB) to maintain the stability of the BAC
• has selectable markers for antibiotic resistance, and phage promoters for transcription of inserted genes

How do BACs work? First, short segments of the organism’s DNA (approximately 100,000 to 300,000 base pairs) are inserted into the BACs. The BAC DNA is amplified as the bacterial cells that have taken up the BACs grow and divide. The cloned BAC DNA can then be isolated, purified, and sequenced. Using computer simulation, the sequenced parts are then rearranged to reveal the genomic sequence of the organism.

Prior to the creation of BACs, yeast artificial chromosomes (YACs) were used for the Human Genome Project. While YACs are capable of holding up to 1,000 kb of DNA and can be used to express eukaryotic proteins that require posttranslational modification, their extremely fragile nature prompted researchers to discontinue their use in favor of BACs.

Moreover, YACs have a high incidence of chimerism (up to 50% in some cases). This creates serious problems, since the presence of these artifacts lead to inconsistent results and errors in interpretation.

The Importance of BACs in Genome Sequencing

• Their ability to hold larger pieces of DNA significantly reduced the number of clones needed to cover the entire human genome.
• BACs are ideal for physically mapping genomes, since they are stable in culture and are easy to manipulate.
• They contain the necessary promoter, enhancer, and silencer required to mimic the natural expression of the gene of interest.
• The imprinting capacity of BACs can be examined outside the normal chromosomal context.
• BACs can be used in studying the developmental consequence of the expression of single genes.
• They can be modified (i.e., insert, delete, or alter sequences) to address mechanistic and functional questions.
• BACs can be modified in vitro in less than 4 weeks.

Currently, BACs are utilized in studying neurological diseases such as Alzheimer’s disease and abnormal chromosomal conditions like Down syndrome. They are also used in studying oncogenes as well as DNA and RNA viruses that include the coronaviruses, herpesviruses, and poxviruses.