Subcloning for DNA Sequencing

  • Gary M. Studnicka
  • Shau-Ping Lei
  • Hun-Chi Lin
  • Gary Wilcox
Part of the Methods in Molecular Biology™ book series (MIMB, volume 23)

Abstract

Chemical (1) and enzymatic (2) methods for determining DNA sequences have revolutionized the techniques of molecular genetics and subsequently our understanding of the gene. The use of singlestranded Ml3 bacteriophage (3–5), in conjunction with Sanger’s dideoxy chain-termination sequencing procedure (2), has greatly increased the rate at which genes can be analyzed. One prerequisite for an efficient sequencing strategy is a set of subclones whose endpoints are evenly distributed along the entire sequence. Shotgun subcloning methods ((6),(7)) require rigorous fractionation of the DNA fragments before cloning, to insure appropriately sized subclones in the final library. Since clones are then chosen at random from the library, shotgun methods are very inefficient at completing the last few small sequence gaps that remain near the end of the project. Various nonrandom subcloning methods have also been developed, utilizing partial restriction digests (8), BAL-3 1 digestion (9), exonuclease III digestion (l0–13), and DNase I (14).

Keywords

Phenol Albumin EDTA Agarose Bromide 

References

  1. 1.
    Maxam, A. M and Gilbert, W. (1977) A new method for sequencing DNA Proc Natl. Acad Sci. USA 74, 560–564.PubMedCrossRefGoogle Scholar
  2. 2.
    Sanger, F., Nicklen, S., and Coulson, A. R(1977) DNA sequencing with chainterminating inhibitors.Proc. Natl. Acad. Set. USA 74, 5463–5467.CrossRefGoogle Scholar
  3. 3.
    Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA sequencing. Nucl. Acids Res. 9, 309–321.PubMedCrossRefGoogle Scholar
  4. 4.
    Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal prtmers. Gene 19, 259–268PubMedCrossRefGoogle Scholar
  5. 5.
    Messmg, J. and Vieira, J (1982) A new pair of Ml3 vectors for selecting either DNA strand of double-digest restriction fragments Gene 19, 269–276.CrossRefGoogle Scholar
  6. 6.
    Anderson, S (1981) Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucl Aads Res 9, 3015–3027.CrossRefGoogle Scholar
  7. 7.
    Deininger, P. L (1983) Approaches to rapid DNA sequence analysis. Anal.Biochem 129, 216–223PubMedCrossRefGoogle Scholar
  8. 8.
    Lamperti, E. D. and Villa-Komaroff, L. (1990) Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases. Anal. Biochem. 185, 187–193.PubMedCrossRefGoogle Scholar
  9. 9.
    Poncz, M., Solowiejczyk, D., Ballantine, M., Schwartz, E, and Surrey, S. (1982)“Nonrandom” DNA sequence analysis in bacteriophage Ml3 by the dideoxy chain-termination method. Proc. Natl. Acad See. USA 79, 4298–4302CrossRefGoogle Scholar
  10. 10.
    Guo, L. H. and Wu, R. (1982) New rapid methods for DNA sequencing based on exonuclease III digestion followed by repair synthesis Nucl. Acids Res 10, 2065–2084.PubMedCrossRefGoogle Scholar
  11. 11.
    Henikoff, S. (1984) Umdirectional dtgestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351–359.PubMedCrossRefGoogle Scholar
  12. 12.
    Henikoff, S (1990) Ordered deletions for DNA sequencing and m vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates Nucl. Acids Res. 18, 2961–2966PubMedCrossRefGoogle Scholar
  13. 13.
    Gronostajski, R. M and Sadowski, P. D (1985) Determmatton of DNA sequences essential for FLP mediated recombination by a novel method J Biol. Chem. 260, 12,32O–12,327.Google Scholar
  14. 14.
    Hong, G. F (1982) A systematic DNA sequencing strategy.J Mol. Biol. 158, 539–549PubMedCrossRefGoogle Scholar
  15. 15.
    Lm, H.-C., Lei, S.-P., and Wilcox, G. (1985) An improved DNA sequencmg strategy Anal. Biochem. 147, 114–119.CrossRefGoogle Scholar
  16. 16.
    Ish-Horowitz, D., and Burke, J. F. (1981) Rapid and efficient cosmtd clonmg Nuci Acids Res. 9, 2989–2998.CrossRefGoogle Scholar
  17. 17.
    Rigby, P W J., Dieckmann, M, Rhodes, C, and Berg, P. (1977) Labelmg deoxyribonucletc acid to high specific activity by nick translation with DNA polymerase I. J Mol. Biof. 113, 237–251CrossRefGoogle Scholar
  18. 18.
    Lis, J T. (1980) Fractionatton of DNA fragments by polyethylene glycol induced precrpitation. Meth. Enzymol 65, 347–353PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. Totowa, New Jersey 1993

Authors and Affiliations

  • Gary M. Studnicka
    • 1
  • Shau-Ping Lei
    • 1
  • Hun-Chi Lin
    • 1
  • Gary Wilcox
    • 1
  1. 1.XOMASanta Monica

Personalised recommendations