Multicopy vectors, such as ColEl derived plasmids, are valuable because they give higher yields of DNA than single copy vectors. However, they also have disadvantages. In particular, the inserts may be unstable especially if they are very long and contain repeated sequences. Many times, unstable inserts are deleted from the plasmid by recombination events. Eukaryotic DNA is particularly unstable in plasmids. Therefore, cloning large segments of eukaryotic DNA in bacteria is now done using bacterial artificial chromosomes (BACs). These are single copy vectors based on the F-plasmid of E. coli. They can accept inserts of 300 kb or more. Electroporation is necessary to trans-
bacterial artificial chromosome (BAC) Single copy vector based on the F-plasmid of E. coli that can carry very long inserts of DNA. Widely used in the human genome project yeast artificial chromosome (YAC) Single copy vector based on yeast chromosome that can carry very long inserts of DNA. Widely used in the human genome project
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FIGURE 22.22 Yeast Artificial Chromosome (YAC)
The YAC has two forms, a circular form for growing in bacteria, and a linear form for growing in yeast. The circular form can be manipulated and grown like any other plasmid in bacteria since it has a bacterial origin of replication and an antibiotic resistance gene. In order to use this in yeast, the circular form is isolated and linearized such that the yeast telomere sequences are on each end. This form can accommodate up to 2,000 kb of cloned DNA inserted into its multiple cloning site (MCS).
form these large constructs into E. coli host cells and the yields of DNA are low. Nonetheless bacterial artificial chromosomes have been widely used in the human genome project and other eukaryotic genome sequencing projects.
Another cloning vector used for larger eukaryotic DNA segments is the P1 artificial chromosome (PAC). This cloning vector is derived from bacteriophage P1, and has been used to carry inserts of up to 150 kb. Just like the lambda derived vectors (see above), these PACs require in vitro packaging. Artificial chromosomes based on P1 have also been made for use in E. coli host cells.
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