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

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.

Evolving a Peptide: Library Platforms and Diversification Strategies

“…If a larger fragment is to be cloned, the "megaprimer" approach is applied by amplification with a series of oligonucleotides [254]. This method can also benefit from using "spiked" synthetic oligonucleotides, allowing randomization at multiple sites [255,256]. Cassette mutagenesis is based on Kunkel mutagenesis, which is time-consuming, so researchers developed an improved version termed ''PFunkel", a conflation of Pfu DNA polymerase and Kunkel mutagenesis, that can be performed in a day's work [257,258].…”

“…Avoiding incorporation of stop codons is practically unattainable and the system is inclined towards amino acid residues encoded by redundant codons [261]. This problem can be tackled by adjusting the mutational frequency with "spiked oligonucleotides" [255], taking into account the differences in reactivity of mononucleotides and the redundant genetic code. The essence of DNA spiking is that non-equimolar ratio of bases at targeted positions are applied during oligonucleotide synthesis, meaning each wild type nucleotide can be custom "doped", achieving either "soft" (high incidence of a certain nucleotide) or "hard" (equal incidence of all four nucleotides) randomization, manually tuning the occurrence of certain amino acids at defined positions in the (poly)peptide chain.…”

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

“…By manipulating DNA amplification conditions, ideal mismatches can be created due to the nature of the polymerase that lacks 3′-5′ proofreading ability. A commercially available Taq polymerase, Mutazyme (Agilent Technologies), was engineered for error-prone PCR to reduce its mutational bias for prohibitive selection preference on certain nucleotides during amplification [131,132]. Meanwhile, MutaGen™ is an in vitro random mutagenesis approach that utilizes low-fidelity human DNA polymerase, known as mutases [133].…”

“…This approach is very much similar to that used for the construction of the HuCAL library. With respect to large-scale mutagenesis, this method can be optimized by using spiked synthetic oligonucleotides that carry different mutations, allowing randomization of one or several cassettes in any given region of the target gene [132,214]. The conventional cassette mutagenesis relies on Kunkel mutagenesis, which is very time consuming, while a recent improved Kunkel methodology, named PFunkel mutagenesis [215], enables mutagenesis to be performed in just one day [160].…”

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

“…Thus, the result is a rather large library, however, with only a small number of potentially successful candidates. 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 ].…”