A “Step Down” PCR-Based Technique for Walking Into and the Subsequent Direct-Sequence Analysis of Flanking Genomic DNA

  • Ziguo Zhang
  • Sarah Jane Gurr
Part of the Methods in Molecular Biology™ book series (MIMB, volume 192)


The hunt for missing sequence data, whether it be in pursuit of full-length clones or for promoter sequences, can be laborious and expensive. Indeed, protracted efforts to find missing sequences by library screening can be fraught with the frustration of foraging through libraries that may lack the relevant clones. Such problems have led several researchers to describe alternative methods to clone and analyze DNA adjacent to known sequences.


Polymerase Chain Reaction Polymerase Chain Reaction Amplification Polymerase Chain Reaction Protocol Adaptor Ligation Magnesium Acetate 
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  1. 1.
    Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.PubMedGoogle Scholar
  2. 2.
    Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16, 8186.CrossRefPubMedGoogle Scholar
  3. 3.
    Parker, J. D., Rabinovitch, P.S., and Burmer, G. C. (1991) Targeted gene walking poly-merase chain reaction. Nucl. Acids Res. 19, 3055–3060.CrossRefPubMedGoogle Scholar
  4. 4.
    Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., Anand, R., Smith, J. C.,and Markham, A. F. (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clone. Nucl. Acids Res. 18, 2887–2890.CrossRefPubMedGoogle Scholar
  5. 5.
    Rosenthal, A. and Jones, D. S. (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucl. Acids Res. 18, 3095–3096.CrossRefPubMedGoogle Scholar
  6. 6.
    Lagerstrom, M., Parik, J., Malmgren, H., Stewart, J., Pettersson, U., and Landegren, U. (1991) Capture PCR: efficient amplification of DAN fragments adjacent to a known sequence in human and Yac DNA. PCR Meth. Appl. 1, 111–119.Google Scholar
  7. 7.
    Jones, D. H.and Winistorfer, S. C. (1993) A method for the amplification of unknown flanking DNA: targeted inverted repeat amplification. Biotechniques 15, 894–904.PubMedGoogle Scholar
  8. 8.
    Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A., and Lukyanov, S. A. (1995) An improved PCR method for walking in uncloned genomic DNA. Nucl. Acids Res. 23, 1087–1088.CrossRefPubMedGoogle Scholar
  9. 9.
    Lukyanov, S. A., Gurskaya, N. G., Lukyanov, K. A., Tarabykin, V. S., and Sverdlov, E. D. (1994) Highly efficient subtractive hybridization of cDNA. Bioorgan. Chem. 20, 701–704.Google Scholar
  10. 10.
    Padegimas, L. S. and Reichert, N. A. (1998) Adaptor ligation-based polymerase chain reaction-mediated walking. Analyt. Biochem. 260, 149–153.CrossRefPubMedGoogle Scholar
  11. 11.
    Weber, K. L., Bolander, M. E., and Sarkab, G. (1998) Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR.Biotechniques 25, 415–419.PubMedGoogle Scholar
  12. 12.
    Cormack, R. S. and Somssich, I. E. (1997) Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA. Gene 194, 273–276.CrossRefPubMedGoogle Scholar
  13. 13.
    Rudi, K., Fossheim, T., and Jakobsen, K. S. (1999) Restriction cutting independent method for cloning genomic DNA segments outside the boundaries of known sequences.Biotechniques 27, 1170–1172, 1176-1177.PubMedGoogle Scholar
  14. 14.
    Zhang, Z and Gurr, S. J. (2000) Walking into the unknown: a “step down” PCR-based technique leading to the direct sequence analysis of flanking genomic DNA. Gene 253, 145–150.CrossRefPubMedGoogle Scholar
  15. 15.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Deidman, J. G., and Strahl, K. (Eds.), Current Protocols on Molecular Biology, 1987. Wiley, NY, 233.Google Scholar
  16. 16.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY.Google Scholar
  17. 17.
    Zhang, Z. and Gurr, S. J. (2001) Expression and sequence analysis of the Blumeria graminis mitogen-activated protein kinase genes, mpk1 and mpk2. Gene 266, 57–65.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Ziguo Zhang
    • 1
  • Sarah Jane Gurr
    • 1
  1. 1.Department of Plant SciencesUniversity of OxfordOxfordUK

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