Regional mutagenesis using Dissociation in maize
Regional mutagenesis using Dissociation in maize
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DOI：
10.1016/j.ymeth.2009.04.009
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发表时间：
2009-11-01
期刊：
影响因子：
4.8
通讯作者：
Brutnell, Thomas P.
中科院分区：
文献类型：
--
作者：
Ahern, Kevin R.;Deewatthanawong, Prasit;Brutnell, Thomas P.
我们描述了利用解离(DS)作为玉米插入诱变剂的遗传筛选、分子方法和最新可用的网络资源。超过1700个DS元素分布在整个玉米基因组中，作为局部或区域诱变的供体元素。描述了两种用于识别感兴趣基因(GOI)中DS插入的基因筛查。在方案I中，当隐性参考等位基因可用时，DS用于产生插入等位基因。DS插入将使目标基因的克隆成为可能，并可用于创建等位基因系列。在方案II中，使用基于PCR的筛查来鉴定GOI中的DS插入，以在测交后代群体中鉴定罕见的插入等位基因。我们详细介绍了一种快速扩增DS插入等位基因两侧序列的反向PCR方法，并描述了一种高通量的96孔板DNA提取方法，用于从幼苗组织中提取高质量的基因组DNA。我们还描述了几种基于网络的工具，用于浏览、搜索和访问所描述的遗传材料。这些DS插入系的建立有望极大地加速玉米功能基因组学的研究。(C)2009 Elsevier Inc.保留所有权利。
We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation (Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize. (C) 2009 Elsevier Inc. All rights reserved.