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The fosmid system has been widely used in the construction of large-insert genomic libraries. Fosmid clones are similar in insert size comparing with cosmid clones and smaller than that in BAC and yeast artificial chromosomes (YAC). Based on E. coli F-plasmids, the single copy of fosmid clones exists in the host bacteria, and maintains high stability through 160 generations of monoclonal subculture. Fosmid vectors contain both a single-copy origin and a high-copy oriV replication origin, which can be induced to produce a high number of copies of the plasmid (approximately 50 copies). There is no potential for bias in inserts, because cloning begins with sheared DNA lead to highly random generation of fragments in contrast to the conventional approach of generating DNA fragments by partial restriction endonuclease digestion. Furthermore, the inserts are flanked with specific sequencing primers that facilitate subsequent sequencing.
(1) Isolation of genomic DNA
Prepare high-molecular-weight genomic DNA from the organism using the Creative Biogene DNA Purification Kit or standard methods. Resuspend the DNA in TE buffer at a concentration of 0.5 μg/μl.
(2) Amplification of genomic DNA
The purified DNA is subjected to randomly primed RCA to amplify any DNA present in the water samples, according to the procedure detailed in the Creative Biogene DNA Amplification Kit manual. The amplified genomic DNA is precipitated by ethanol and then dissolved in 10 mM Tris-HCl (pH 7.5) and 1 mM ethylenediaminetetraacetic acid. Most of the random primers and dNTPs are removed after precipitation with ethanol.
(3) Fosmid library construction
The amplified high-molecular-weight DNA is cloned with fosmid vectors. In order to select DNA fragments longer than 20 kb, the amplified DNA is visualized by pulsed-field gel electrophoresis (PFGE). And DNA fragments larger than 20 kb are cut out, extract from the gel, and re-run for 5 h under the same conditions. These resolved DNA fragments are used to construct a fosmid library.
The ligated DNA is then packaged and is plated on phage T1-resistant Escherichia coli cells to produce the library. Packaging a single 10-μL ligation reaction generates more than 100,000 fosmid clones from the DNA isolated. DNA from several randomly chosen clones is purified for high-throughput fosmid sequencing.
(4) Size estimation of Fosmid clone
To evaluate the average insert size in the library, some clones are randomly selected from the library. The plasmid DNA samples from these clones are completely digested with Not I, and the insert sizes are estimated via 1% agarose gel electrophoresis at 90 V overnight. The λDNA marker (48.5 kb) and D15000 marker are used to estimate the size of insert sequence.
(5) DNA sequencing
Each colony from the different libraries is picked and then inoculated in Luria Broth media containing chloramphenicol and glycerol (10%, final concentration) in a 384-well plate. The cells are cultured at 37℃ for 1 day and stored at -80℃. DNA can be isolated using the Kit according to the instructions. Finally, the clones from each library are selected for the sequencing reaction. BLAST sequence homology searches are conducted against the GenBank database after subtracting the vector sequence.