Assembling DNA fragments by USER fusion.
Assembling DNA fragments by USER fusion.
复制标题
DOI：
10.1007/978-1-61779-564-0_7
复制
发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Chandrasegaran, Srinivasan
中科院分区：
文献类型：
--
作者：
Annaluru, Narayana;Muller, Heloise;Chandrasegaran, Srinivasan
DNA合成技术的最新进展使设计和合成几个KB大小的DNA片段成为可能。但是，将较小的DNA片段组装到较大的DNA段中的过程仍然是一个繁琐的过程。在本章中，我们描述了尿嘧啶特异性切除反应（用户）介导的方法在体外和体内（使用大肠杆菌）快速有效地组装多个DNA片段。对于用户融合体外组件，使用含有单个尿嘧啶（U）和DNA聚合酶的适当前向和反向引物对每个单个构件（BBS）（BBS）（BBS）（BBS）（将要组装）进行了0.75 kb（要组装）。相邻BBS之间的重叠为8-13个碱基对。将扩增的BB的等摩尔混合在一起，并由用户酶处理，以在相邻的BBS之间产生互补的3'单链悬垂，然后将其连接并同时放大以产生较大的3-KB段。然后将组装的片段克隆到质粒载体中，并测序以确认其身份。对于大肠杆菌中的用户融合体内组件，在存在合成质粒的情况下，对BBS进行了用户处理，该质粒在5'bb的5'末端和3''的5'末端和3''的情况下进行了8-13个碱基对重叠。 - 混合物中3'bb的末端。然后将用户处理的产品直接转换为大肠杆菌，以有效并正确重建包含所需目标插入物的重组质粒。后一种方法还用于将三个不同的靶基因迅速组装到载体中，以形成新的合成质粒构建体。
Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific excision reaction (USER)-mediated approach for rapid and efficient assembly of multiple DNA fragments both in vitro and in vivo (using Escherichia coli). For USER fusion in vitro assembly, each of the individual building blocks (BBs), 0.75 kb in size (that are to be assembled), was amplified using the appropriate forward and reverse primers containing a single uracil (U) and DNA polymerase. The overlaps between adjoining BBs were 8-13 base pairs. An equimolar of the amplified BBs were mixed together and treated by USER enzymes to generate complementary 3' single-strand overhangs between adjoining BBs, which were then ligated and amplified simultaneously to generate the larger 3-kb segments. The assembled fragments were then cloned into plasmid vectors and sequenced to confirm their identity. For USER fusion in vivo assembly in E. coli, USER treatment of the BBs was performed in the presence of a synthetic plasmid, which had 8-13 base pair overlaps at the 5'-end of the 5' BB and at the 3'-end of the 3' BB in the mixture. The USER treated product was then transformed directly into E. coli to efficiently and correctly reconstitute the recombinant plasmid containing the desired target insert. The latter approach was also used to rapidly assemble three different target genes into a vector to form a new synthetic plasmid construct.