Economical analysis of saturation mutagenesis experiments
Economical analysis of saturation mutagenesis experiments
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DOI：
10.1038/srep10654
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发表时间：
2015-07-20
影响因子：
4.6
通讯作者：
Nov, Yuval
中科院分区：
文献类型：
--
作者：
Acevedo-Rocha, Carlos G.;Reetz, Manfred T.;Nov, Yuval
饱和诱变是一种强大的蛋白质、代谢途径和基因组工程技术。尽管它有许多应用，但创建高质量的饱和诱变文库仍然是一个挑战，因为各种实验参数以复杂的方式影响所产生的多样性。我们从经济学的角度探讨了饱和诱变文库制备的各个方面：我们引入了一种更便宜、更快速的基于液体介质的文库质量评估方法；分析了在有和没有冗余的文库中，引物纯度和供应商的作用；使用基于诱变剂混合的传统和紧急随机化方案，比较了文库质量、产量、随机化效率和退火偏倚；并建立了一种在给定筛选成本和其他实验参数的情况下选择最具成本效益的随机化方案的方法。我们表明，通过仔细考虑这些参数，可以显著降低实验室费用。
Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.