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DNA Sequencing Protocols pp 13–31Cite as

The Universal Primers and the Shotgun DNA Sequencing Method

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 167))

Abstract

For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in Escherichia coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques such as ultracentrifugation unnecessary. Although today the polymerase chain reaction (PCR) is a valuable alternative for the amplification of small DNA pieces (1), it cannot substitute for the construction of libraries of DNA fragments. Therefore, E. coli has served not only as a vehicle to amplify DNA, but also to separate many DNA molecules of similar length and the two DNA strands simultaneously. For this purpose, a bacteriophage such as M13 can be used. The various viral cis- and trans-acting functions are critical not only for strand separation, but also to separate the single-stranded DNA from the E. coli cell by an active transport mechanism through the intact cell wall.

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References

  1. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Ehrlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 293, 487–491.

    Article  Google Scholar 

  2. Scott, J. and Smith, G. (1990) Searching for peptide ligands with an epitope library. Science 249, 386–390.

    Article  PubMed  CAS  Google Scholar 

  3. Edgell, M. H., Hutchison, C. A., III, and Sclair, M. (1972) Specific endonuclease R fragments of bacteriophage φFX174 deoxyribonucleic acid. J. Virol. 9, 574–582.

    PubMed  CAS  Google Scholar 

  4. Beaucage, S. L. and Caruthers, M. H. (1981) Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859–1862.

    Article  CAS  Google Scholar 

  5. Hutchison, C. A., III, Phillips, S., Edgell, M. H., Gillam, S., Jahnke, P., and Smith, M. (1978) Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253, 6551–6560.

    PubMed  CAS  Google Scholar 

  6. Smith, M., Leung, D. W., Gillam, S., Astell, C. R., Montgomery, D. L., and Hall, B. D. (1979) Sequence of the gene for iso-1-cyto-chrome C in Saccharomyces cerevisiae. Cell 16, 753–761.

    CAS  Google Scholar 

  7. Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–268.

    Article  PubMed  CAS  Google Scholar 

  8. Zoller, M. and Smith, M. (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 10, 6487–6500.

    Article  PubMed  CAS  Google Scholar 

  9. Norrander, J., Kempe, T., and Messing, J. (1983) Improved M13 vectors using oligonucleotide-directed mutagenesis. Gene 26, 101–106.

    Article  PubMed  CAS  Google Scholar 

  10. Sanger, F., Donelson, J. E., Coulson, A. R., Kössel, H., and Fischer, H. (1973) Use of DNA polymerase I primed by a synthetic oligonucleotide to determine a nucleotide sequence in phage f1 DNA. Proc. Natl. Acad. Sci. USA 70, 1209–1213.

    Article  PubMed  CAS  Google Scholar 

  11. Hofschneider, P. H. (1963) Untersuchungen üuber “kleine” E. coli K12 Bacteriophagen M12, M13, und M20. Z. Naturforschg. 18b, 203–205.

    CAS  Google Scholar 

  12. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., et al. (1995). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512.

    Article  PubMed  CAS  Google Scholar 

  13. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. V., Heynecker, H. L., Boyer, H. W., Crosa, J. W., and Falkow, S. (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95–113.

    Article  PubMed  CAS  Google Scholar 

  14. Vovis, G. F. and Ohsumi, M. (1978) The filamentous phages as transducing particles, in The Single-Stranded DNA Phages (Denhardt, D. T., Dressler, D., and Ray, D. S., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445–448.

    Google Scholar 

  15. Herrmann, R., Neugebauer, K., Zentgraf, H., and Schaller, H. (1978) Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd. Mol. Gen. Genet. 159, 171.

    Article  PubMed  CAS  Google Scholar 

  16. Salivar, W. O., Henry, T. J., and Pratt, D. (1967) Purification and properties of diploid particles of coliphageM13. Virology 32, 41–51.

    Article  PubMed  CAS  Google Scholar 

  17. Mandel, M. and Higa, A. (1970) Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159–162.

    Article  PubMed  CAS  Google Scholar 

  18. Malamy, M. H., Fiandt, M., and Szybalski, W. (1972) Electron microscopy of polar insertions in the lac operon of Escherichia coli. Mol. Gen. Genet. 119, 207–222.

    CAS  Google Scholar 

  19. Landy, A., Olchowski, E., and Ross, W. (1974) Isolation of a functional lac regulatory region. Mol. Gen. Genet. 133, 273–281.

    Article  PubMed  CAS  Google Scholar 

  20. Messing, J. (1991) Cloning in M13 phage or how to use biology at its best. Gene, 100, 3–12.

    Article  PubMed  CAS  Google Scholar 

  21. Messing, J., Gronenborn, B., Müller-Hill, B., and Hofschneider, P. H. (1977) Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in the M1 3 replicative form in vitro. Proc. Natl. Acad. Sci. USA 74, 3642–3646.

    Article  PubMed  CAS  Google Scholar 

  22. Messing, J. and Gronenborn, B. (1978) The filamentous phage M13 as carrier DNA for operon fusions in vitro, in The Single-Stranded DNA Phages (Denhardt, D. T., Dressler, D., and Ray, D. S., eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 449–453.

    Google Scholar 

  23. Gronenborn, B. and Messing, J. (1978) Methylation of single-stranded DNA in vitro introduces new restrictions endonuclease cleavage sites. Nature 272, 375–377.

    Article  PubMed  CAS  Google Scholar 

  24. Dotto, G. P. and Zinder, N. D. (1984) Reduction of the minimal sequence for initiation of DNA synthesis by qualitative and quantitative changes of an initiator protein. Nature 311, 279–280.

    Article  PubMed  CAS  Google Scholar 

  25. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.

    Article  PubMed  CAS  Google Scholar 

  26. Heidecker, G., Messing, J., and Gronenborn, B. (1980) A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene 10, 69–73.

    Article  PubMed  CAS  Google Scholar 

  27. Messing, J. (1979) A multipurpose cloning system based on the single-stranded DNA bacteriophage M13. Recombinant DNA Tech. Bull. NIH Publication No. 79-99, 2, No. 2, 43–48.

    CAS  Google Scholar 

  28. Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA sequencing. Nucleic Acids Res. 9, 309–321.

    Article  PubMed  CAS  Google Scholar 

  29. Gardner, R. C., Howarth, A. J., Hahn, P. O., Brown-Leudi, M., Shepherd, R. J., and Messing, J. (1981) The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shot-gun sequencing. Nucleic Acids Res. 9, 287l–2888.

    Article  Google Scholar 

  30. Holden, C. (1991) Briefings. Science 254, 28.

    Google Scholar 

  31. Messing, J. (1983) New M13 vectors for cloning. Methods Enzymol. 101, 20–78.

    Article  PubMed  CAS  Google Scholar 

  32. Larson, R. and Messing, J. (1982) Apple II software for M13 shotgun DNA sequencing. Nucleic Acids Res. 10, 39–49.

    Article  PubMed  CAS  Google Scholar 

  33. Larson, R. and Messing, J. (1983) Apple II computer software for DNA and protein sequence data. DNA 2, 31–35.

    Article  PubMed  CAS  Google Scholar 

  34. Hackett, P. H., Fuchs, J. A., and Messing, J. (1984) An introduction to recombinant DNA techniques, in Basic Experiments in Gene Manipulation, Benjamin-Cummings, Menlo Park, CA.

    Google Scholar 

  35. Messing, J., Vieira, J., and Gardner, R. (1982) Codon insertion mutagenesis to study functional domains of β-lactamase, in In Vitro Mutagenesis, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 52.

    Google Scholar 

  36. Messing, J. and Seeburg, P. H. (1981) A strategy for high speed DNA sequencing, in Developmental Biology Using Purified Genes (Brown, D. and Fox, F., eds.), ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. XXIII. Academic, NY, pp. 659–669.

    Google Scholar 

  37. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp and pUC vectors. Gene 33, 103–119.

    Article  PubMed  CAS  Google Scholar 

  38. Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, CR., Heiner, C., Kent, S. B., and Hood, L. E. (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321, 674–679.

    Article  PubMed  CAS  Google Scholar 

  39. Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencing with fluorescent chainterminating dideoxynucleotides. Science 238, 336–341.

    Article  PubMed  CAS  Google Scholar 

  40. Ewing, B. Hillier, L. Wendl, M. C., and Green, P. (1998) Base-calling of automated sequencer traces using phred-I-accuracy assessment. Genome Research 8, 175–185.

    PubMed  CAS  Google Scholar 

  41. Ewing, B. and Green, P. (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Research 8, 186–194.

    PubMed  CAS  Google Scholar 

  42. Osoegawa, K., Woon, P-Y, Zhao, B., Frengen, E., Tateno, M., Catanese, J. J., and de Jong, P. J. (1998) An improved approach for construction of bacterial artificial chromosome libraries. Genomics 52, 1–8.

    Article  PubMed  CAS  Google Scholar 

  43. Venter, J. C., Smith, H. O., and Hood, L. (1996) A new strategy for genome sequencing. Nature 381, 364–366.

    Article  PubMed  CAS  Google Scholar 

  44. Venter, J. C., Adams, M. D., Sutton, G. G., Kerlavage, A. R., Smith, H. O., and Hunkapiller, M. (1998) Shotgun sequencing of the human genome. Science 280, 1540–1542.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Waksman Institute, Rutgers

    Joachim Messing

  2. State University of NJ, Piscataway, NJ

    Joachim Messing

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  1. Joachim Messing
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Editor information

Editors and Affiliations

  1. Northern Ireland Regional Genetics Centre, Belfast City Hospital Trust, Northern Ireland, UK

    Colin A. Graham

  2. Department of Anatomy and Physiology, University of Dundee, Scotland, UK

    Alison J. M. Hill

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© 2001 Humana Press Inc.

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Messing, J. (2001). The Universal Primers and the Shotgun DNA Sequencing Method. In: Graham, C.A., Hill, A.J.M. (eds) DNA Sequencing Protocols. Methods in Molecular Biology™, vol 167. Humana Press. https://doi.org/10.1385/1-59259-113-2:013

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  • DOI: https://doi.org/10.1385/1-59259-113-2:013

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-716-8

  • Online ISBN: 978-1-59259-113-8

  • eBook Packages: Springer Protocols

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