BCA Library Construction Protocol
DNA cloning, especially large DNA cloning, is the first step in contemporary complex genome analysis. Cloning technology of high-molecular-weight DNA has been developed mainly using Escherichia coli and yeast as hosts. In the early stages of the Human Genome Project, yeast artificial chromosome (YAC) libraries have been generated and used for constructing the framework of genome. The YAC cloning system has a great advantage of cloning of very large DNA (>500 kb), thereby facilitating construction of a physical map of the complex genome. The bacterial artificial chromosome (BAC) technologies matured later but proved to have so many advantages that the BAC libraries have been the primary input to contig assembly and the public sector human genome sequencing.
BACs are easily purified as plasmid DNAs, have little if any chimerism, and are stable, with a very few interesting exceptions. BAC uses the E. coli F-factor plasmid replication to maintain largeness (100–350 kb). Genomic DNA is subjected to partial digestion with a restriction endonuclease to break DNA into clonable size and size fractionated using pulsed field gel electrophoresis (PFGE). The size-fractionated DNA is cloned into a BAC vector and transformed into E. coli by electrical shock. The transformants are arrayed into microtiter dishes and high-density replica filters are prepared to facilitate screening of the library.
The Workflow of BCA Library Construction
- Firstly, you 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.
- Then, these isolated cells are mixed with hot agarose. The whole mixture is poured into a mould to produce a set of small blocks, each containing thousands of the isolated cells.
- These cells are treated with enzymes to dissolve their cell membranes and release the DNA into the agarose gel.
- Construction of a BAC library requires generation of relatively high molecular weight restriction fragments (100 kb - 350 kb). Such restriction fragments will serve as inserts in BAC construction. In order to obtain fragments in this size range, the high molecular weight DNA in the agarose plugs must be partially digested with a restriction enzyme. To determine the conditions that yield a maximum percentage of fragments between 100 and 350 kb, a series of partial restriction digests is performed.
- Once the optimal conditions for producing fragments between 100 and 300 kb are determined, a mass of digestion using several plugs is performed. The partially digested DNA from these plugs then can be used as insert DNA in construction of a BAC library.
- Then, these blocks of gel containing chopped up DNA are inserted into holes in a slab of agarose gel. The DNA fragments are separated according to size by electrophoresis.
- A solution of ‘markers’ is inserted on the other side of the gel. These are DNA fragments of known size that 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.
- These DNA fragments are inserted into a BAC vector using a ligase enzyme to join the two bits of DNA together. Next, the BAC clones are added to bacterial cells, usually E. coli.
- The bacteria are then spread on nutrient-rich plates which allow only the bacteria that carry BAC clones to grow. The bacteria grow rapidly, producing lots of bacterial cells, each containing a copy of the BAC clone.
- Then, the bacteria are ‘picked’ into plates of 96 or 384 so that each tube contains a single BAC clone. The bacteria can be copied or frozen and kept until researchers are ready to use the DNA for sequencing.
For research use only. Not intended for any clinical use.