Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Mar 11;21(5):1239–1244. doi: 10.1093/nar/21.5.1239

Exoquence DNA sequencing.

C Li 1, P W Tucker 1
PMCID: PMC309288  PMID: 8464708

Abstract

We have developed a strategy for DNA sequencing based on exonuclease III digestion followed by double strand specific endonuclease digestion and direct dideoxynucleotide sequencing reaction. This strategy eliminates the need for subcloning, oligonucleotide primers, and prior knowledge of the DNA to be sequenced. All template and primer duplexes needed for sequencing a complete insert can be prepared in one day from uncharacterized starting DNA. Sequence information can be obtained from different regions of the DNA simultaneously. The method uses double-stranded DNA to generate single-stranded template and primer, and thus produces high quality sequence results. Commercially available dideoxy-sequencing kits are well suited for this method. The strategy should be applicable for both automatic and routine laboratory DNA sequencing.

Full text

PDF
1243

Images in this article

1241Image
on p.1241
1242Image
on p.1242
1242Image
on p.1242

Selected References

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

  1. Carothers A. M., Urlaub G., Mucha J., Grunberger D., Chasin L. A. Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method. Biotechniques. 1989 May;7(5):494-6, 498-9. [PubMed] [Google Scholar]
  2. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Church G. M., Kieffer-Higgins S. Multiplex DNA sequencing. Science. 1988 Apr 8;240(4849):185–188. doi: 10.1126/science.3353714. [DOI] [PubMed] [Google Scholar]
  4. Donelson J. E., Wu R. Nucleotide sequence analysis of deoxyribonucleic acid. VII. Characterization of Escherichia coli exonuclease 3 activity for possible use in terminal nucleotide sequence analysis of duplex deoxyribonucleic acid. J Biol Chem. 1972 Jul 25;247(14):4661–4668. [PubMed] [Google Scholar]
  5. Engelke D. R., Hoener P. A., Collins F. S. Direct sequencing of enzymatically amplified human genomic DNA. Proc Natl Acad Sci U S A. 1988 Jan;85(2):544–548. doi: 10.1073/pnas.85.2.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guo L. H., Wu R. Exonuclease III: use for DNA sequence analysis and in specific deletions of nucleotides. Methods Enzymol. 1983;100:60–96. doi: 10.1016/0076-6879(83)00046-4. [DOI] [PubMed] [Google Scholar]
  7. Guo L. H., Wu R. New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis. Nucleic Acids Res. 1982 Mar 25;10(6):2065–2084. doi: 10.1093/nar/10.6.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  9. Hunkapiller T., Kaiser R. J., Koop B. F., Hood L. Large-scale and automated DNA sequence determination. Science. 1991 Oct 4;254(5028):59–67. doi: 10.1126/science.1925562. [DOI] [PubMed] [Google Scholar]
  10. Innis M. A., Myambo K. B., Gelfand D. H., Brow M. A. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. doi: 10.1073/pnas.85.24.9436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Putney S. D., Benkovic S. J., Schimmel P. R. A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7350–7354. doi: 10.1073/pnas.78.12.7350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. RICHARDSON C. C., LEHMAN I. R., KORNBERG A. A DEOXYRIBONUCLEIC ACID PHOSPHATASE-EXONUCLEASE FROM ESCHERICHIA COLI. II. CHARACTERIZATION OF THE EXONUCLEASE ACTIVITY. J Biol Chem. 1964 Jan;239:251–258. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Sorge J. A., Blinderman L. A. ExoMeth sequencing of DNA: eliminating the need for subcloning and oligonucleotide primers. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9208–9212. doi: 10.1073/pnas.86.23.9208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wu R., Ruben G., Siegel B., Jay E., Spielman P., Tu C. P. Synchronous digestion of SV40 DNA by exonuclease III. Biochemistry. 1976 Feb 24;15(4):734–740. doi: 10.1021/bi00649a003. [DOI] [PubMed] [Google Scholar]
  19. Zimmermann J., Voss H., Schwager C., Stegemann J., Ansorge W. Automated Sanger dideoxy sequencing reaction protocol. FEBS Lett. 1988 Jun 20;233(2):432–436. doi: 10.1016/0014-5793(88)80477-0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES