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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1057)

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 

References

  1. 1.
    Benito MI, Walbot V (1997) Characterization of the maize mutator transposable element MURA transposase as a DNA-binding protein. Mol Cell Biol 17:5165–5175PubMedGoogle Scholar
  2. 2.
    Devon RS, Porteous DJ, Brookes AJ (1995) Splinkerettes-improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res 23:1644–1645PubMedCrossRefGoogle Scholar
  3. 3.
    Edwards D et al (2002) Amplification and detection of transposon insertion flanking sequences using fluorescent muAFLP. Biotechniques 32:1090–1092, 1094, 1096–1097Google Scholar
  4. 4.
    Frey M, Stettner C, Gierl A (1998) A general method for gene isolation in tagging approaches: amplification of insertion mutagenised sites (AIMS). Plant J 13:717–821CrossRefGoogle Scholar
  5. 5.
    Hengen PN (1995) Vectorette, splinkerette and boomerang DNA amplification. Trends Biochem Sci 20:372–373PubMedCrossRefGoogle Scholar
  6. 6.
    Jones DH, Winistorfer SC (1992) Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA. Nucleic Acids Res 20:595–600PubMedCrossRefGoogle Scholar
  7. 7.
    Kilstrup M, Kristiansen KN (2000) Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer. Nucleic Acids Res 28:E55PubMedCrossRefGoogle Scholar
  8. 8.
    O'Malley R et al (2007) An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat Protoc 2:2910–2917PubMedCrossRefGoogle Scholar
  9. 9.
    Riley J et al (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res 18:2887–2890PubMedCrossRefGoogle Scholar
  10. 10.
    Uren AG et al (2009) A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat Protoc 4:789–798PubMedCrossRefGoogle Scholar
  11. 11.
    Settles AM, Latshaw S, McCarty DR (2004) Molecular analysis of high-copy insertion sites in maize. Nucleic Acids Res 32:e54PubMedCrossRefGoogle Scholar
  12. 12.
    Walbot V, Warren C (1988) Regulation of Mu element copy number in maize lines with an active or inactive mutator transposable element system. Mol Gen Genet 211:27–34PubMedCrossRefGoogle Scholar
  13. 13.
    Liu S, Dietrich CR, Schnable PS (2009) DLA-based strategies for cloning insertion mutants: cloning the gl4 locus of maize using Mu transposon tagged alleles. Genetics 183:1215–1225PubMedCrossRefGoogle Scholar
  14. 14.
    Liu S et al (2009) Mu transposon insertion sites and meiotic recombination events co-localize with epigenetic marks for open chromatin across the maize genome. PLoS Genet 5:e1000733PubMedCrossRefGoogle Scholar
  15. 15.
    Yunis I, Salazar M, Yunis EJ (1991) HLA-DR generic typing by AFLP. Tissue Antigens 38: 78–88Google Scholar
  16. 16.
    Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88:9828–9832Google Scholar
  17. 17.
    Qiu F et al (2003) DNA sequence-based "bar codes" for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources. Plant Physiol 133:475–481PubMedCrossRefGoogle Scholar

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

Personalised recommendations