Skip to main content
SpringerLink
Log in

Chromosomal walking of flanking regions from short known sequences in GC-rich plant genomic DNA

  • Protocols
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

High-efficiency thermal asymmetric interlaced (HE-TAIL) PCR is a modified thermal asymmetric interlaced (TAIL) method for finding unknown genomic DNA sequences adjacent to known sequences in GC-rich plant DNA. Necessary modifications to obtain high-efficiency amplification of flanking sequences are the inclusion of 2 control reactions during tertiary cycling and the design of long gene-specific primers, which can be used during single-step annealing-extension PCR. The modified protocol is suitable to walk from short known sequences, such as sequence-tagged sites (STS), expressed sequence tags (EST), or short exon sequences, and enables researchers to clone full-length open reading frames (ORFs) without library screening. Moreover, the HE-TAIL method can be used to identify DNA sequences flanking T-DNA insertions or to isolate promoter regions. Although individual steps are limited to about 4 kb, multiple steps can be done to walk upstream or downstream of known regions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

EtBr:

ethidium bromide

HE-TAIL:

high-efficiency thermal asymmetric interlaced

References

  • Cottage A, Yang A, Maunders H, Lacy C, and Ramsay N (2001) Identification of DNA sequences flanking T-DNA insertions by PCR-walking. Plant Mol Biol Rep 19: 321–327.

    Article  CAS  Google Scholar 

  • Dabo SM, Mitchell ED, and Melcher U (1993) A method for isolation of nuclear DNA from cotton leaves. Anal Biochem 210: 34–38.

    Article  PubMed  CAS  Google Scholar 

  • Dempster EL, Pryor KV, Francis D, Young JE, and Rogers HJ (1999) Rapid DNA extraction from Ferns for PCR based analysis. BioTechniques 27: 66–68.

    PubMed  CAS  Google Scholar 

  • Liu Y, Mitsukawa N, Oosumi T, and Whittier RF (1995) Efficient isolation and mapping ofArabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8: 457–463.

    Article  PubMed  CAS  Google Scholar 

  • Liu Y and Whittier R (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674–681.

    Article  PubMed  CAS  Google Scholar 

  • Mazars GR, Moyret C, Jeanteur P, and Theillet CG (1991) Direct sequencing by thermal asymmetric PCR. Nucl Acids Res 19: 4783.

    Article  PubMed  CAS  Google Scholar 

  • Michiels A, Van den Ende W, Tucker M, Van Riet L, and Van Laere A (2003) Extraction of high-quality genomic DNA from latex-containing plants. Anal Biochem 315: 85–89.

    Article  PubMed  CAS  Google Scholar 

  • Nakazaki T, Ihara N, Fucuta Y, and Ikehashi H (2000) Abundant polymorphism in the flanking regions of two loci for basic PR-1 proteins as markers forIndica japonica differentiation in rice. Breeding Sci 50: 173–181.

    Article  CAS  Google Scholar 

  • Nakazaki T and Ikehashi H (1998) Genomic sequence and polymorphisms of a rice chitinase gene, Cht4. Breeding Sci 48: 371–376.

    CAS  Google Scholar 

  • Ochman H, Gerber A, and Hartl D (1988) Genetic application of an inverse polymerase chain reaction. Genetics 120: 621–623.

    PubMed  CAS  Google Scholar 

  • Rosenthal A and Jones D (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucl Acid Res 18: 3095–3096.

    Article  CAS  Google Scholar 

  • Sato Y, Murakami T, Funatsuki H, Matsuba S, Saruyama H, and Tanida M (2001) Heat shock mediated APX gene expression and protection against chilling injury in rice seedlings. J Exp Bot 52: 145–151.

    Article  PubMed  CAS  Google Scholar 

  • Siebert P, Chenchik A, Kellog D, Lukyanov K, and Lukyanov S (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acid Res 23: 1087–1088.

    Article  PubMed  CAS  Google Scholar 

  • Terauchi R and Kahl G (2000) Rapid isolation of promoter sequences by TAIL-PCR: the 5′ flanking regions of Pal and Pgi genes from yams (Dioscorea). Mol Gen Genet 263: 554–560.

    Article  PubMed  CAS  Google Scholar 

  • Van den Ende W, Michiels A, Van Wonterghem D, Clerens S, De Roover J, and Van Laere A (2001) Defoliation induces 1-FEHII (Fructan 1-exohydrolase II) in witloof chicory roots. Cloning and purification two isoforms (1-FEH IIa and 1-FEH IIb). Mass fingerprint of the 1-FEH II enzymes. Plant Physiol 126: 1186–1195.

    Article  PubMed  Google Scholar 

  • Woodhead M, Davies HV, Brennan RM, and Taylor MA (1998) The isolation of genomic DNA from Blackcurrant (Ribes nigrum L.). Mol Biotech 9: 243–246.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. An Michiels

    Present address: Department of Cell Biology and Molecular genetics, H.J. Patterson Hall, University of Maryland, 20742-5815, College Park, MD, USA

Authors and Affiliations

  1. Laboratory of Developmental Biology, Institute of Botany and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001, Leuven, Belgium

    An Michiels, Wim van den Ende & Andre Van Laere

  2. USDA/ARS/SGIL, Building 6, BARC-West, 10300 Baltimore Avenue, 20705-2350, Beltsville, MD, USA

    Mark Tucker

Authors
  1. An Michiels
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark Tucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wim van den Ende
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andre Van Laere
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to An Michiels.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Michiels, A., Tucker, M., van den Ende, W. et al. Chromosomal walking of flanking regions from short known sequences in GC-rich plant genomic DNA. Plant Mol Biol Rep 21, 295–302 (2003). https://doi.org/10.1007/BF02772805

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02772805

Key words

Search

Navigation