Ferrin L J
Division of Gastroenterology, MMC 36 University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, USA.
Mol Biotechnol. 2001 Jul;18(3):233-41. doi: 10.1385/MB:18:3:233.
With the explosion in genetic information and almost complete sequencing of the human genome, a shift in the experimental goals of molecular biologists is occurring. Instead of focusing on single genes, current attempts seek to divine the interactions of several genes and sequences. This requires increasingly complex genetic constructs and manipulations, often of very large DNA constructs, and these can be made with RecA protein-based techniques. When RecA protein combined with an oligonucleotide acts as a sequence-specific "masking tape" to block DNA from the action of DNA modifying enzymes, and can be used to direct the cleavage of DNA at single predetermined restriction endonuclease sites. This reaction is called RecA-Assisted Restriction Endonuclease (RARE) Cleavage. The reverse reaction, known as RecA-Assisted Ligation, can be used to join any two desired fragments. When one of those fragments is a vector, a desired fragment can be cloned directly without constructing a genomic library. The reagents and equipment needed are relatively inexpensive, and almost any desired genetic construct up to about 300 kb in size can be made in a straightforward manner.
随着遗传信息的激增以及人类基因组几乎完全测序，分子生物学家的实验目标正在发生转变。当前的研究不再聚焦于单个基因，而是试图探寻多个基因和序列之间的相互作用。这需要越来越复杂的基因构建体和操作，通常涉及非常大的DNA构建体，而这些可以通过基于RecA蛋白的技术来实现。当RecA蛋白与寡核苷酸结合时，它会作为一种序列特异性的“胶带”，阻止DNA受到DNA修饰酶的作用，并可用于在单个预定的限制性内切酶位点引导DNA的切割。这个反应被称为RecA辅助限制性内切酶（RARE）切割。其逆反应，即RecA辅助连接，可用于连接任何两个所需的片段。当其中一个片段是载体时，无需构建基因组文库就可以直接克隆所需的片段。所需的试剂和设备相对便宜，几乎任何大小约为300 kb的所需基因构建体都可以以直接的方式制备。
