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We have demonstrated that saturation mutagenesis libraries with no codon redundancy and amino acid biases could be easily constructed by our newly introduced small-intelligent strategy. Since the only difference between small-intelligent primer and the conventional NNS primer is the codon composition, it is reasonable to assume that any PCR strategies that work with NNS primer are adoptable with small-intelligent primer. Thus, the small-intelligent strategy is feasible for the construction of high-quality saturation libraries and could serve as a good substitute for NNS randomization.
In comparison with the MAX system, the small-intelligent system offers an alternative for producing libraries with the same quality as MAX does, but are less complex and less expensive in most cases, since there are no requirements for ligation, restriction digestion, as well as template synthesis, and fewer oligonucleotides are needed in the small-intelligent system relative to the MAX system, except for the case where multiple contiguous sites are randomized. Most importantly, our system would allow for randomization of multiple contiguous amino acids, although more oligonucleotides would need to be synthesized. However, this situation could be improved by applying the iterative CASTing method (23).
In addition to full randomization, the small-intelligent system should also allow us to perform any restricted randomizations using a specific primer set, and such primer sets could be easily achieved with the assistance of DC-Analyzer. Taken together, the small-intelligent strategy could serve as a simple and valuable tool to construct high-quality focused mutagenesis libraries.
This research was supported by the National Natural Science Foundation of China (no. 20872014).
The authors declare no competing interests.
Address correspondence to Lixia Tang, School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu 610054, China. e-mail: [email protected]
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