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Plant Transposable Elements

Volume 1057 of the series Methods in Molecular Biology pp 167-176

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Digestion–Ligation–Amplification (DLA): A Simple Genome Walking Method to Amplify Unknown Sequences Flanking Mutator (Mu) Transposons and Thereby Facilitate Gene Cloning

  • Sanzhen LiuAffiliated withIowa State University
  • , An-Ping HsiaAffiliated withIowa State University
  • , Patrick S. SchnableAffiliated withCenter for Plant Genomics, Roy J Carver Co-Lab, Iowa State University

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Abstract

Digestion–ligation–amplification (DLA), a novel PCR-based genome walking method, was developed to amplify unknown sequences flanking known sequences of interest. DLA specifically overcomes the problems associated with amplifying genomic sequences flanking high copy number transposons in large genomes. Two DLA-based strategies, MuClone and DLA-454, were developed to isolate Mu-tagged alleles. MuClone allows for the amplification of DNA flanking subsets of the numerous Mu transposons in the genome using unique three-nucleotide tags at the 3′-ends of primers, simplifying the identification of flanking sequences that co-segregate with mutant phenotypes caused by Mu insertions. DLA-454, which combines DLA with 454 pyrosequencing, permits the efficient amplification and sequencing of Mu flanking regions in a high-throughput manner.

Key words

Genome walking Ligation-mediated PCR Next-gen sequencing Gene cloning Mu transposon Mutator