RF cloning:: A restriction-free method for inserting target genes into plasmids
RF cloning:: A restriction-free method for inserting target genes into plasmids
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DOI：
10.1016/j.jbbm.2005.12.008
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发表时间：
2006-04-30
影响因子：
--
通讯作者：
Löwe, J
中科院分区：
文献类型：
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作者：
van den Ent, F;Löwe, J
无限制克隆（RF）提供了一种简单、通用的方法，可以精确地将DNA片段插入到圆形质粒内的任何所需位置，而不受限制位点、连接或载体或感兴趣基因的改变的影响。该技术使用编码感兴趣基因的PCR片段作为一对引物，在环状质粒周围进行线性扩增反应。与QuickChange （TM）定点诱变（引入单个突变或小的插入/缺失）不同，RF克隆插入完整的基因而不引入多余的残基。该蛋白没有任何改变，引物设计和程序本身都很简单，这使得它适合于高通量表达，是结构基因组学的理想选择。英国皇家版权所有(c) 2006出版由爱思唯尔B.V.。
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 (TM) 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. Crown Copyright (c) 2006 Published by Elsevier B.V. All rights reserved.