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- Title
DLA-Based Strategies for Cloning Insertion Mutants: Cloning the gl4 Locus of Maize Using Mit Transposon Tagged Alleles.
- Authors
Sanzhen Liu; Dietricht, Charles R.; Schnable, Patrick S.
- Abstract
Digestion-ligation-amplification (DLA), a novel adaptor-mediated PCR-based method that uses a single-stranded oligo as the adaptor, was developed to overcome difficulties of amplifying unknown sequences flanking known DNA sequences in large genomes. DLA specifically overcomes the problems associated with existing methods for amplifying genomic sequences flanking Mu transposons, including high levels of nonspecific amplification. Two DLA-based strategies, MuClone and DLA-454, were developed to isolate Mu-tagged alleles. MuClorie allows for the amplification of subsets of the numerous Mu transposons in the genome, using unique three-nucleotide tags at the 3λ ends of primers, simplifying the identification of flanking sequences that cosegregate with mutant phenotypes caused by Mu insertions. DLA-454, which combines DLA with 454 pyrosequencing, permits the efficient cloning of genes for which multiple independent insertion alleles are available without the need to develop segregating populations. The utility of each approach was validated by independently cloning the gl4 (glossy4) gene. Mutants of gl4 lack the normal accumulation of epicuticular waxes. The gl4 gene is a homolog of the Arabidopsis CUT1 gene, which encodes a condensing enzyme involved in the synthesis of very-long-chain fatty acids, which are precursors of epicuticular waxes.
- Subjects
GENETIC engineering of corn; PLANT genetic engineering; PLANT genetics; PLANT mutation; CLONING; POLYMERASE chain reaction; NUCLEOTIDE sequence
- Publication
Genetics, 2009, Vol 183, Issue 4, p1215
- ISSN
0016-6731
- Publication type
Article
- DOI
10.1534/genetics.109.108936