A high-throughput Arabidopsis reverse genetics system
A high-throughput Arabidopsis reverse genetics system
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DOI：
10.1105/tpc.004630
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发表时间：
2002-12-01
期刊：
影响因子：
11.6
通讯作者：
Goff, SA
中科院分区：
文献类型：
--
作者：
Sessions, A;Burke, E;Goff, SA
为了提高功能基因组学的效率，我们收集了已知位点有T-DNA插入的拟南芥系。开发了一种高通量改良的热不对称交错(TAIL)-PCR协议，并用于扩增来自近100,000个转化系的T-DNA左边界两侧的DNA片段。对来自52,964个T-DNA系的85,108个TAIL-PCR产物进行测序，并与拟南芥基因组进行比较，以确定T-DNA在每个系中的位置。当绘制预测的T-DNA插入位点时，显示出与预测的编码序列的偏差。拟南芥基因索引（Arabidopsis Gene Index）名称搜索或BLAST （Basic Local Alignment Search Tool）搜索可识别目标基因的预期插入突变。插入可以通过单个品系的简单PCR检测来确认。预测插入在340个测试品系中有257个（76%）得到确认。该资源被命名为SAIL（先正达拟南芥插入库），科学界可在www.tmri.org上获得。
A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from similar to100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.