Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis genome
Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis genome
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DOI：
10.1046/j.1365-313x.1998.00146.x
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发表时间：
1998-05-01
期刊：
影响因子：
7.2
通讯作者：
Micol, JL
中科院分区：
文献类型：
--
作者：
Ponce, MR;Quesada, V;Micol, JL
提出了对先前用于恢复拟南芥基因组DNA侧翼T-DNA插入的方法的改进，其允许避免由T-DNA插入的多联体性质引起的一些克隆困难。该程序的原理是通过大小对突变DNA的限制性片段进行分类，这些片段是用Ndel和Bst 1107 I在单独的双酶切中产生的。鉴于这两种酶的位点在pGV 3850：1003 T-DNA构建体中是连续的，所获得的限制性片段分为两类：在两个双端序列中显示相同大小的那些，其对应于T-DNA多联体内部的序列;以及不同大小的那些，其包含植物DNA和T-DNA插入物之间的连接。这样的标准使得有可能容易地区分对应于内部T-DNA部分的消化产物，其不值得进一步关注，以及那些可能包括感兴趣的基因座的片段的消化产物。基因组突变体DNA的限制性片段之间的区分可以在拯救的质粒、反向PCR扩增产物或基因组印迹中的条带上进行。
An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with Ndel and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.