Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly

ACS Omega
Edson CárcamoPaul Gaytán

Abstract

In vitro mutagenesis methods have revolutionized biological research and the biotechnology industry. In this study, we describe a mutagenesis method based on synthesizing a gene using a complete set of forward and reverse spiked oligonucleotides that have been modified to introduce a low ratio of mutant nucleotides at each position. This novel mutagenesis scheme named "Spiked Genes" yields a library of clones with an enhanced mutation distribution due to its unbiased nucleotide incorporation. Using the far-red fluorescent protein emKate as a model, we demonstrated that Spiked Genes yields richer libraries than those obtained via enzymatic methods. We obtained a library without bias toward any nucleotide or base pair and with even mutations, transitions, and transversion frequencies. Compared with enzymatic methods, the proposed synthetic approach for the creation of gene libraries represents an improved strategy for screening protein variants and does not require a starting template.

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Citations

Jan 2, 2020·International Journal of Molecular Sciences·Krištof Bozovičar, Tomaž Bratkovič
Apr 18, 2019·International Journal of Molecular Sciences·Chia Chiu LimTheam Soon Lim
Mar 10, 2018·Beilstein Journal of Organic Chemistry·Ruth SuchslandSabine Müller
May 1, 2021·Biology Methods and Protocols·Shreya RouthRiddhiman Dhar
Jun 3, 2021·Viruses·Thomas D Burton, Nicholas S Eyre

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Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

Diversify
GeneMorph II
GeneMorph

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