Oligonucleotide-directed random mutagenesis using a high-efficiency procedure

Dale, Susan; Belfield, Maxine; Richardson, Thomas

doi:10.1016/s1046-2023(05)80167-7

Cited by 4 publications

(4 citation statements)

References 7 publications

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“…This work confirmed the robustness of the method to produce all of the expected mutations by sequencing 546 clones and finding 88 different base substitutions out of the 90 possible single mutants. A similar mutagenesis approach was reported the following year by Hill et al and 4 years later by Dale et al Using genes as templates, researchers incorporated spiked oligonucleotides by PCR-based methods to modify specific regions of the encoded proteins . More recently, Hidalgo et al and Jin et al reused the spiked oligonucleotides to achieve a focused and directed evolution in certain regions of the regulatory elements and enzymes via in vitro recombination with oligonucleotides bearing the wild-type sequence.…”

Section: Introductionmentioning

confidence: 94%

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

et al. 2017

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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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Section: Introductionmentioning

confidence: 94%

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

et al. 2017

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“…Applying a simple formula, the volume of spiking mixture required for a desired number of average replacements per oligonucleotide can be easily calculated: 33 V aliquots of spiking mix ¼ error rate Â 1:33 Â V pure building blocks…”

Section: Spiked Oligonucleotidesmentioning

confidence: 99%

“…For further generation of gene libraries, the prepared randomized oligos usually are used as primers in PCR amplification followed by assembly to the full lengths gene segment and cloning into an appropriate vector. 29,30,33 Over the past years, a host of methods has been developed allowing for efficient cloning, as for example the recently reported one-pot methodology for cassette randomisation and recombination including megaprimer PCR. 3 For an overview of cloning strategies the reader is referred to references.…”

Section: Final Preparation Of Gene Librariesmentioning

confidence: 99%

Mixed oligonucleotides for random mutagenesis: best way of making them

et al. 2012

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The generation of proteins, especially enzymes, with pre-deliberated, novel properties is a big challenge in the field of protein engineering. This aim, over the years was critically facilitated by newly emerging methods of combinatorial and evolutionary techniques, such as combinatorial gene synthesis followed by functional screening of many structural variants generated in parallel (library). Libraries can be generated by a large number of available methods. Therein the use of mixtures of pre-formed trinucleotide blocks representing codons for the 20 canonical amino acids for oligonucleotide synthesis stands out as allowing fully controlled partial (or total) randomization individually at any number of arbitrarily chosen codon positions of a given gene. This has created substantial demand of fully protected trinucleotide synthons of good reactivity in standard oligonucleotide synthesis. We here review methods for the preparation of oligonucleotide mixtures with a strong focus on codon-specific trinucleotide blocks.

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“…There are strategies to at least partially circumvent this problem, like using NNS instead of NNN codons (with N = A, C, G, T; S = C, G) taking advantage of redundancy of the third nucleotide positions in the majority of codons [ 10 ], or using spiked oligonucleotides [ 11 ], which are synthesized from solutions of the four nucleotide building blocks, each of those contaminated with a "spiking mix" consisting of equal aliquots of each of the four building blocks [ 9 , 12 ]. The required volume of the spiking mix to achieve a desired amount of nucleotide replacements at a defined position of the oligonucleotide can be calculated, such that library size and degree of randomization can be restricted [ 13 – 14 ]. Nevertheless, although those methods and sophisticated variations of them [ 14 – 17 ] have improved library design and synthesis, full control over randomization is not possible.…”

Section: Introductionmentioning

confidence: 99%

Preparation of trinucleotide phosphoramidites as synthons for the synthesis of gene libraries

Suchsland¹,

Appel²,

Müller³

2018

Beilstein J. Org. Chem.

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The preparation of protein libraries is a key issue in protein engineering and biotechnology. Such libraries can be prepared by a variety of methods, starting from the respective gene library. The challenge in gene library preparation is to achieve controlled total or partial randomization at any predefined number and position of codons of a given gene, in order to obtain a library with a maximum number of potentially successful candidates. This purpose is best achieved by the usage of trinucleotide synthons for codon-based gene synthesis. We here review the strategies for the preparation of fully protected trinucleotides, emphasizing more recent developments for their synthesis on solid phase and on soluble polymers, and their use as synthons in standard DNA synthesis.

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Oligonucleotide-directed random mutagenesis using a high-efficiency procedure

Cited by 4 publications

References 7 publications

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

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

Mixed oligonucleotides for random mutagenesis: best way of making them

Preparation of trinucleotide phosphoramidites as synthons for the synthesis of gene libraries

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