An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome
An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome
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DOI：
10.1038/nprot.2007.425
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Ecker, Joseph R.
中科院分区：
文献类型：
--
作者：
O'Malley, Ronan C.;Alonso, Jose M.;Ecker, Joseph R.
农杆菌转移DNA（AgrobacteriumtransferDNA，T-DNA）是一种有效的植物诱变剂，已被用于在拟南芥中建立序列索引的T-DNA插入系，作为研究基因功能的工具。创建T-DNA插入系需要一种可靠的方法来定位基因组中的插入位点。在该方案中，我们描述了一种接头连接介导的PCR方法，我们已经使用该方法筛选突变体文库并鉴定了超过150，000个T-DNA插入突变体;该方法也可以应用于绘制单个突变体。该程序由三个步骤组成：限制性酶介导的衔接子与基因组DNA的连接;用对衔接子和T-DNA特异性的引物对T-DNA/基因组DNA连接点进行PCR扩增;以及对T-DNA/基因组连接点进行测序，以能够映射到参考基因组。在大多数情况下，测序的基因组区域延伸到T-DNA边界，使得能够鉴定插入物的确切位置。整个过程需要两周时间才能完成。
Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.