Abstract
We have developed a novel method to clone and sequence minute quantities of DNA. The method was applied to sequence a 180 kb plasmid pNL1. The first step was the production of a size distributed population of DNA molecules that were derived from the 180 kb plasmid pNL1. The first step was accomplished by a random synthesis reaction using Klenow fragment and random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN6, 5'-GTAATACGACTCACTATAGGGCNNNNNN-3'. In the second step, Klenow-synthesized molecules were amplified by PCR using T7 primer (5'-GTAATACGACTCACTATAGGGC-3'). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules with sizes ranging from 28 bp to >23 kb which were detectable on an agarose gel. The Klenow-synthesized molecules were then used as templates for standard PCR with T7 primer. PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for cloning and sequencing. From the same Klenow-synthesized molecules, we were also able to generate PCR products with sizes up to 23 kb by long range PCR. A total 232.5 kb sequences were obtained from 593 plasmid clones and over twenty putative genes were identified. Sequences from these 593 clones were assembled into 62 contigs and 99 individual sequence fragments with a total unique sequence of 86.3 kb.
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- Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
- Fredrickson J. K., Balkwill D. L., Drake G. R., Romine M. F., Ringelberg D. B., White D. C. Aromatic-degrading Sphingomonas isolates from the deep subsurface. Appl Environ Microbiol. 1995 May;61(5):1917–1922. doi: 10.1128/aem.61.5.1917-1922.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fredrickson J. K., Brockman F. J., Workman D. J., Li S. W., Stevens T. O. Isolation and characterization of a subsurface bacterium capable of growth on toluene, naphthalene, and other aromatic compounds. Appl Environ Microbiol. 1991 Mar;57(3):796–803. doi: 10.1128/aem.57.3.796-803.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grothues D., Cantor C. R., Smith C. L. PCR amplification of megabase DNA with tagged random primers (T-PCR). Nucleic Acids Res. 1993 Mar 11;21(5):1321–1322. doi: 10.1093/nar/21.5.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kinzler K. W., Vogelstein B. Whole genome PCR: application to the identification of sequences bound by gene regulatory proteins. Nucleic Acids Res. 1989 May 25;17(10):3645–3653. doi: 10.1093/nar/17.10.3645. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stillwell L. C., Thurston S. J., Schneider R. P., Romine M. F., Fredrickson J. K., Saffer J. D. Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199. J Bacteriol. 1995 Aug;177(15):4537–4539. doi: 10.1128/jb.177.15.4537-4539.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sun F., Arnheim N., Waterman M. S. Whole genome amplification of single cells: mathematical analysis of PEP and tagged PCR. Nucleic Acids Res. 1995 Aug 11;23(15):3034–3040. doi: 10.1093/nar/23.15.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang L., Cui X., Schmitt K., Hubert R., Navidi W., Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847–5851. doi: 10.1073/pnas.89.13.5847. [DOI] [PMC free article] [PubMed] [Google Scholar]