Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs
Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs
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DOI：
10.1038/nbt0102-76
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发表时间：
2002-01-01
影响因子：
46.9
通讯作者：
Sisido, M
中科院分区：
文献类型：
--
作者：
Murakami, H;Hohsaka, T;Sisido, M
建立了突变基因文库的一般构建方法。该方法称为随机插入/缺失（RID）诱变，能够在随机位置缺失任意数量的连续碱基，同时将任意数量的特定序列或随机序列插入相同位置。通过用GFP（uv）基因中的Bg/II识别序列（AGATCT）替换三个随机选择的连续碱基来证明RID诱变的适用性。此外，随机选择的三个碱基被20个密码子的混合物取代。这些突变体在大肠杆菌中表达，并选择显示出与野生型GFP不同的荧光性质的突变体。在所选的6个突变体中，发现了一个黄色荧光蛋白和一个增强的绿色荧光蛋白，这两个蛋白都不能通过易错PCR诱变获得。还获得了显示不同荧光性质的DsRed蛋白的几种突变体。
A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the Bg/ll recognition sequence (AGATCT) in the GFP(uv) gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.