Large culture volumes are required to obtain sufficient quantities of BAC DNA for analysis, leading to a high degree of RNA contamination. Life Technologies Inc. has also developed BAC-specific protocols for its Concert High Purity Plasmid Purification Systems. Product purity is touted as equivalent to that obtained by double banding on CsCl gradients. Prep 96 Kits can either be used with the QIAvac manifold or automated with the BioRobot 9600 multipurpose workstation. Samples are processed by gentle alkaline lysis in four to six hours with a yield similar to the kits.
MACs replicate and segregate as natural chromosomes.
A linear TAR cloning vector is basically a YAC vector flanked by a small amount of sequence from the target gene on one end and a human repetitive Alu sequence on the other end. When this linear TAR cloning vector is transformed into yeast along with human genomic DNA, recombination results in generation of YAC clones containing the human gene insert. TAR vectors also contain F-factor sequences for transfer to E. coli as BACs. Powerful yeast genetics, our ability to manipulate bacterial genes, and the growing knowledge of human genetics have created a small universe of YACs, PACs, BACs, MACs, and a growing number of satellite molecular techniques. The use of these chromosome-like constructs for complex gene sequencing, gene product characterization, and eventually gene therapeutics, will keep many people busy for years to come.