The CopyControl™ Fosmid Library Production Kits* provide an efficient and improved method for constructing a library of approximately 40 kb clones. The CopyControl pCC1FOS™ Vector contains both the E. coli F-factor single-copy origin of replication and the inducible high-copy oriV (Fig. 1). CopyControl Fosmid clones are typically grown at single copy to ensure insert stability and successful cloning of encoded and expressed toxic protein and unstable DNA sequences. The CopyControl Fosmid clones can then be induced up to 50 copies per cell immediately before DNA purification. This step greatly increases DNA yields, while maintaining the stability of the plasmid.
The CopyControl HTP Fosmid Library contains the pCC2FOS™ Vector which is designed to optimize end-sequencing results, especially in a high-throughput setting. The primer cassette, engineered in conjunction with Agencourt Bioscience Corporation, eliminates wasteful and expensive vector sequence reads by having the 3´ ends of the primer-annealing sites only three bases from the vector/insert junction. In addition, the seven-base sequence at the 3´ end of each primer was specifically designed to minimize mispriming to any contaminating E. coli DNA present after template purification (Fig. 2).
The kit uses a strategy of cloning blunt-ended DNA fragments generated by random shearing of the DNA, to produce more complete and unbiased genomic libraries than can be obtained by partial restriction endonuclease digests. Genomic DNA is first sheared into approximately 40-kb fragments. The sheared DNA is end-repaired to generate blunt, 5´-phosphorylated ends and then size-selected by and recovered from a low-melting-point agarose gel. Finally, the size-selected DNA is ligated into the cloning-ready CopyControl pCC1FOS or pCC2FOS Vector, packaged using ultra-high efficiency MaxPlax™ Lambda Packaging Extracts (>109 pfu/µg DNA), included in the kit, and plated on the supplied TransforMax™ EPI300™ E. coli (Fig. 3).
Figure 1. CopyControl™ Vector map. The CopyControl pCC1FOS™ and pCC2FOS™ Vectors for CopyControl Fosmid library production are supplied linearized at the Eco72 I (blunt) site and then dephosphorylated. The vector is ready for cloning end-repaired (blunt-end) genomic DNA of approximately 40 kb.
Figure 2. The CopyControl™ pCC2FOS™ Vector primer cassette. The vector differs from the pCC1FOS Vector by the engineering of a new primer cassette that eliminates wasteful vector-derived sequencing reads and minimizes the potential for priming on the E. coli genome.
Figure 3 (click to enlarge). Overview of the process for preparing a fosmid library using the CopyControl™ Fosmid Library Production Kits. Once the library has been prepared, individual clones can be cultured in small volume and induced to multiple-copy number for high yields of high-purity DNA for fingerprinting, sequencing, etc., using Epicentre's DirectLysis Fosmid96 kit or FosmidMAX™ DNA Purification Kit.
Figure 4. Typical sequencing results obtained with the pCC2FOS™ Forward Primer on a pCC2FOS™ clone at 1/48x BigDye™ dilution. Similar results were obtained with the pCC2FOS Reverse Primer (data not shown).
*Covered by issued and/or pending patents.
|Figure 5. CopyControl™ Fosmid clones can be induced up to 50 copies per cell to greatly increase DNA yield. Hind III digests of fosmid DNA isolated from uninduced (–) and induced (+) CopyControl clones. Digests contained one-third (8 µl) of the total sample volume and were analyzed by agarose gel electrophoresis. Lane M, Kilobase ladder.|