RAPID AND EFFICIENT SITE-SPECIFIC MUTAGENESIS WITHOUT PHENOTYPIC SELECTION
RAPID AND EFFICIENT SITE-SPECIFIC MUTAGENESIS WITHOUT PHENOTYPIC SELECTION
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DOI：
10.1073/pnas.82.2.488
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
KUNKEL, TA
中科院分区：
文献类型：
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作者：
KUNKEL, TA
将几个单碱基取代突变引入lacZ α。基因在克隆载体M13 mp2中，以40-60%的效率，在仅需要转染标准体外诱变反应的未分级产物的快速程序中。在转染前，对DNA进行两次简单的额外处理，产生接近100%的位点特异性突变频率。该方法适用于表型沉默突变，除了那些可以选择。高效率，. simeq。10-通过使用含有几个尿嘧啶残基代替胸腺嘧啶的DNA模板，获得了比使用没有富集程序的现有方法所观察到的高1倍的DNA。该模板对于定点诱变方案典型的体外反应具有正常的编码潜力，但在转染到野生型中时没有生物活性（即，ung+）大肠杆菌宿主细胞。因此，在新合成的不含尿嘧啶的共价闭合环状互补链中存在的所需变化的表达是非常有利的。该程序应用于通过寡核苷酸和易错聚合引入的突变。除了在改变DNA序列方面的实用性外，这种方法还可以用于检查位于基因内定义位置的特定病变的生物学后果。
Several single-base substitution mutations were introduced into the lacZ.alpha. gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, .simeq. 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure was applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.