Liu Sanzhen, Hsia An-Ping, Schnable Patrick S
Iowa State University, Ames, IA, USA.
Methods Mol Biol. 2013;1057:167-76. doi: 10.1007/978-1-62703-568-2_12.
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.
消化-连接-扩增（DLA）是一种基于聚合酶链式反应（PCR）的新型基因组步移方法，旨在扩增感兴趣的已知序列侧翼的未知序列。DLA特别克服了在大型基因组中扩增高拷贝数转座子侧翼基因组序列所带来的问题。开发了两种基于DLA的策略，即MuClone和DLA-454，用于分离Mu标签等位基因。MuClone利用引物3'端独特的三核苷酸标签，扩增基因组中众多Mu转座子亚集侧翼的DNA，简化了与Mu插入导致的突变表型共分离的侧翼序列的鉴定。DLA-454将DLA与454焦磷酸测序相结合，能够以高通量方式高效扩增和测序Mu侧翼区域。
