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Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs

Abstract

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 BglII recognition sequence (AGATCT) in the GFPUV 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.

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Figure 1: A general scheme of the random insertion/deletion (RID) mutagenesis for the construction of a library of mutant genes.
Figure 2: Analysis of the products in steps 1–8 on 8 M urea–4% PAGE (A–C) and on 4% PAGE (D).
Figure 3: Structures of the 5′- and 3′-anchors.
Figure 4: Mutation positions and sequences of several mutants of the GFPUV gene.
Figure 5: Distribution of mutation positions in the GFPUV genes.
Figure 6: Diversity of the fluorescence properties of GFPUV and DsRed mutants obtained by the random substitution by 20 amino acids.

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Acknowledgements

This work was supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No.11102003). The authors thank K. Takakura and. K. Inoguchi of Okayama University of Science for allowing us to use the Hitachi FM-BIO II instrument. H.M. received a Research Fellowship from the Japan Society for Promotion of Science for Young Scientists.

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Correspondence to Masahiko Sisido.

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Murakami, H., Hohsaka, T. & Sisido, M. Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs. Nat Biotechnol 20, 76–81 (2002). https://doi.org/10.1038/nbt0102-76

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