Settles A Mark, Latshaw Susan, McCarty Donald R
University of Florida, PO Box 110690, Gainesville, FL 32611-0690, USA.
Nucleic Acids Res. 2004 Apr 1;32(6):e54. doi: 10.1093/nar/gnh052.
High-copy transposon mutagenesis is an effective tool for creating gene disruptions in maize. In order to molecularly define transposon-induced disruptions on a genome-wide scale, we optimized TAIL-PCR to amplify genomic DNA flanking maize Robertson's Mutator insertions. Sample sequencing from 43 Mutator stocks and the W22 inbred line identified 676 non-redundant insertions, and only a small fraction of the flanking sequences showed significant similarity to maize repetitive sequences. We further designed and tested 79 arbitrary primers to identify 12 primers that amplify all Mutator insertions within a DNA sample at 3.1-fold redundancy. Importantly, the products are of sufficient size to use as substrates or probes for hybridization-based identification of gene disruptions. Our adaptation simplifies previously published TAIL-PCR protocols and should be transferable to other high-copy insertional mutagens.
高拷贝转座子诱变是在玉米中产生基因破坏的有效工具。为了在全基因组范围内从分子水平上定义转座子诱导的破坏，我们优化了热不对称交错PCR（TAIL-PCR）以扩增玉米罗伯逊Mutator插入位点侧翼的基因组DNA。对43个Mutator株系和W22自交系进行样本测序，鉴定出676个非冗余插入，并且只有一小部分侧翼序列与玉米重复序列具有显著相似性。我们进一步设计并测试了79条任意引物，以鉴定出12条引物，这些引物能以3.1倍冗余度扩增DNA样本中的所有Mutator插入。重要的是，产物的大小足以用作基于杂交的基因破坏鉴定的底物或探针。我们的改进简化了先前发表的TAIL-PCR方案，并且应该可以转移到其他高拷贝插入诱变剂上。
