High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines
High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines
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DOI：
10.2144/03356st01
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发表时间：
2003-12-01
期刊：
影响因子：
2.7
通讯作者：
Weisshaar, B
中科院分区：
文献类型：
--
作者：
Strizhov, N;Li, Y;Weisshaar, B
通过生成侧翼序列标签 (FST) 来表征拟南芥突变体，并针对经济、高通量生产进行了优化，创建了一条管道。 T-DNA 诱变拟南芥植物的 GABI-Kat 集合被用作独立转基因品系的来源。该流程包括以 96 孔格式自动提取基因组 DNA、用于扩增 T-DNA 边界附近植物序列的接头连接 PCR 方法、PCR 产物的自动纯化和测序，以及对所得序列文件的计算修剪。通过以下措施显着提高了数据质量：(i) 对接头连接产物进行限制性消化，以减少由游离质粒衍生的片段共扩增引起的琐碎序列；(ii) 为第二个扩增步骤设计接头引物，以增强单个 PCR 片段的选择性生成，甚至来自具有多个 T-DNA 插入的品系。通过包括这些步骤，避免了凝胶纯化，每条线的扩增反应数量从前四个减少到三个，并且产生至少一个 FST 的线的百分比从 66% 增加到 86%。超过 58,000 个 FST 已提交至 GenBank(R)，可在 http://www.mpiz-koeln.mpg.de/GABI-Kat/ 上获取。
A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced front four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank(R) and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.