Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Aug 25;11(16):5521–5540. doi: 10.1093/nar/11.16.5521

An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.

L H Guo, R C Yang, R Wu
PMCID: PMC326295  PMID: 6310503

Abstract

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.

Full text

PDF
5521

Images in this article

5531Image
on p.5531
5532Image
on p.5532
5534Image
on p.5534

Selected References

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

  1. Anderson S. Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucleic Acids Res. 1981 Jul 10;9(13):3015–3027. doi: 10.1093/nar/9.13.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes W. M., Bevan M. Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition. Nucleic Acids Res. 1983 Jan 25;11(2):349–368. doi: 10.1093/nar/11.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Deininger P. L., Jolly D. J., Rubin C. M., Friedmann T., Schmid C. W. Base sequence studies of 300 nucleotide renatured repeated human DNA clones. J Mol Biol. 1981 Sep 5;151(1):17–33. doi: 10.1016/0022-2836(81)90219-9. [DOI] [PubMed] [Google Scholar]
  4. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gardner R. C., Howarth A. J., Hahn P., Brown-Luedi M., Shepherd R. J., Messing J. The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucleic Acids Res. 1981 Jun 25;9(12):2871–2888. doi: 10.1093/nar/9.12.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gronenborn B., Messing J. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature. 1978 Mar 23;272(5651):375–377. doi: 10.1038/272375a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Hartley J. L., Chen K. K., Donelson J. E. A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing. Nucleic Acids Res. 1982 Jul 10;10(13):4009–4025. doi: 10.1093/nar/10.13.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heidecker G., Messing J., Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. doi: 10.1016/0378-1119(80)90145-6. [DOI] [PubMed] [Google Scholar]
  11. Hong G. F. A systemic DNA sequencing strategy. J Mol Biol. 1982 Jul 5;158(3):539–549. doi: 10.1016/0022-2836(82)90213-3. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  15. Poncz M., Solowiejczyk D., Ballantine M., Schwartz E., Surrey S. "Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4298–4302. doi: 10.1073/pnas.79.14.4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rüther U., Koenen M., Otto K., Müller-Hill B. pUR222, a vector for cloning and rapid chemical sequencing of DNA. Nucleic Acids Res. 1981 Aug 25;9(16):4087–4098. doi: 10.1093/nar/9.16.4087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rüther U. pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts. Nucleic Acids Res. 1982 Oct 11;10(19):5765–5772. doi: 10.1093/nar/10.19.5765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Schmid C. W., Jelinek W. R. The Alu family of dispersed repetitive sequences. Science. 1982 Jun 4;216(4550):1065–1070. doi: 10.1126/science.6281889. [DOI] [PubMed] [Google Scholar]
  21. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  22. Wallace R. B., Johnson M. J., Suggs S. V., Miyoshi K., Bhatt R., Itakura K. A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322. Gene. 1981 Dec;16(1-3):21–26. doi: 10.1016/0378-1119(81)90057-3. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Wu R., Taylor E. Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA. J Mol Biol. 1971 May 14;57(3):491–511. doi: 10.1016/0022-2836(71)90105-7. [DOI] [PubMed] [Google Scholar]
  25. Yang R., Lis J., Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. doi: 10.1016/0076-6879(79)68012-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES