van den Ent Fusinita, Löwe Jan
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
J Biochem Biophys Methods. 2006 Apr 30;67(1):67-74. doi: 10.1016/j.jbbm.2005.12.008. Epub 2006 Feb 3.
Restriction-free (RF) cloning provides a simple, universal method to precisely insert a DNA fragment into any desired location within a circular plasmid, independent of restriction sites, ligation, or alterations in either the vector or the gene of interest. The technique uses a PCR fragment encoding a gene of interest as a pair of primers in a linear amplification reaction around a circular plasmid. In contrast to QuickChange site-directed mutagenesis, which introduces single mutations or small insertions/deletions, RF cloning inserts complete genes without the introduction of unwanted extra residues. The absence of any alterations to the protein as well as the simplicity of both the primer design and the procedure itself makes it suitable for high-throughput expression and ideal for structural genomics.
无限制（RF）克隆提供了一种简单通用的方法，可将DNA片段精确插入环状质粒内的任何所需位置，而无需考虑限制酶切位点、连接反应，也无需对载体或目的基因进行改造。该技术使用编码目的基因的PCR片段作为引物，围绕环状质粒进行线性扩增反应。与引入单突变或小插入/缺失的QuickChange定点诱变不同，RF克隆可插入完整基因，不会引入不需要的额外残基。由于不会对蛋白质造成任何改变，且引物设计和操作过程本身都很简单，因此它适用于高通量表达，是结构基因组学的理想选择。
