Reliable method for generating double-stranded DNA vectors containing site-specific base modifications
Reliable method for generating double-stranded DNA vectors containing site-specific base modifications
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DOI：
10.2144/04375st01
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发表时间：
2004-11-01
期刊：
影响因子：
2.7
通讯作者：
Doetsch, PW
中科院分区：
文献类型：
--
作者：
Brégeon, D;Doetsch, PW
所有生物体的细胞都不断暴露在物理和化学试剂中，这些试剂会破坏DNA并改变其基因组的完整性。尽管不同修复途径的效率相对较高，但仍有一些病变存在。当它被复制或转录时。通过DNA和RNA聚合酶的病变旁路已经成为许多研究的主题。然而，在体内转录病变旁路机制的知识是非常有限的，因为没有强大的方法。在这里，我们描述了一个协议的基础上合成的互补链的环状单链DNA分子，它允许生产大量的双链DNA含有病变在转录序列中的特定位置。这样的构建体随后可用于通过RNA聚合酶的体内病变旁路研究，并确定这些事件如何受到细胞遗传背景的影响。
Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in. DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.