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

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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.

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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 

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Correspondence to An Michiels.

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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

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