Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Sep;86(18):6917–6921. doi: 10.1073/pnas.86.18.6917

A strategy for high-volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides.

F W Studier 1
PMCID: PMC297961  PMID: 2780548

Abstract

Direct sequencing of cosmid DNAs using a library of oligonucleotide primers of length 8, 9, or 10 is proposed. The statistics of priming indicate that a primer library sufficient for determining the sequence of the entire human genome (100,000 cosmids) would be small enough to be assembled and managed. Such a library would greatly reduce the cost and effort of high-volume sequencing: primers would be instantly available; the sequence of each cosmid DNA could be determined from a single DNA preparation without the necessity for mapping or subcloning; and, because each primer would be used repeatedly, the cost of primers would become a negligible fraction of other costs. A combination of random and directed priming could determine the sequence of a cosmid DNA in 1.2-1.5 times the minimum number of sequencing reactions required, and completely directed priming would be even more efficient. The success of this strategy requires that a considerable fraction of octamers, nonamers, or decamers be able to prime selectively in double-stranded DNAs 45,000 base pairs (bp) long; initial results indicate that this is likely to be the case. The strategy is not limited to cloned DNAs and would be useful for rapid identification and direct sequencing of viral nucleic acids.

Full text

PDF
6917

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Collins J., Hohn B. Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4242–4246. doi: 10.1073/pnas.75.9.4242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  3. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  4. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Moffatt B. A., Dunn J. J., Studier F. W. Nucleotide sequence of the gene for bacteriophage T7 RNA polymerase. J Mol Biol. 1984 Feb 25;173(2):265–269. doi: 10.1016/0022-2836(84)90194-3. [DOI] [PubMed] [Google Scholar]
  6. Prober J. M., Trainor G. L., Dam R. J., Hobbs F. W., Robertson C. W., Zagursky R. J., Cocuzza A. J., Jensen M. A., Baumeister K. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science. 1987 Oct 16;238(4825):336–341. doi: 10.1126/science.2443975. [DOI] [PubMed] [Google Scholar]
  7. Sanger F., Coulson A. R., Hong G. F., Hill D. F., Petersen G. B. Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol. 1982 Dec 25;162(4):729–773. doi: 10.1016/0022-2836(82)90546-0. [DOI] [PubMed] [Google Scholar]
  8. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Smith L. M., Sanders J. Z., Kaiser R. J., Hughes P., Dodd C., Connell C. R., Heiner C., Kent S. B., Hood L. E. Fluorescence detection in automated DNA sequence analysis. Nature. 1986 Jun 12;321(6071):674–679. doi: 10.1038/321674a0. [DOI] [PubMed] [Google Scholar]
  10. Wilson R. K., Yuen A. S., Clark S. M., Spence C., Arakelian P., Hood L. E. Automation of dideoxynucleotide DNA sequencing reactions using a robotic workstation. Biotechniques. 1988 Sep;6(8):776-7, 781-7. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES