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

  • Sanzhen Liu
  • An-Ping Hsia
  • Patrick S. Schnable
Part of the Methods in Molecular Biology book series (MIMB, volume 1057)


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 


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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Sanzhen Liu
    • 1
  • An-Ping Hsia
    • 1
  • Patrick S. Schnable
    • 2
  1. 1.Iowa State UniversityAmesUSA
  2. 2.Center for Plant Genomics, Roy J Carver Co-LabIowa State UniversityAmesUSA

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