Tn5 in vitro transposition
Tn5 in vitro transposition
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DOI：
10.1074/jbc.273.13.7367
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发表时间：
1998-03-27
影响因子：
4.8
通讯作者：
Reznikoff, WS
中科院分区：
文献类型：
--
作者：
Goryshin, IY;Reznikoff, WS
本文报道了一种有效的Tn 5体外转座系统的开发。该系统的一个关键组成部分是使用超活性突变转座酶。野生型转座酶的失活可能与体内转座频率低有关。体外实验表明：Tn 5转座的大部分步骤所需的大分子仅为转座酶、特异的19 bp Tn 5末端序列和靶DNA，转座酶可能不能与产物DNA自解离，Tn 5转座以保守的“剪切和粘贴”机制进行;而Tn 5从供体骨架的释放涉及在特定末端序列的末端精确切割3 ′和5 ′链。
This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition. The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3' and 5' strands at the ends of the specific end sequences.