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

 Last Update: 2020-11-08
 Source: Internet
 Author: User

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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,
Mu
Clone and DLA-454, were developed to isolate
Mu
-tagged alleles.
Mu
Clone 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.

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