AbstractRandom mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids.

摘要随机诱变，例如易错PCR（epPCR），是一种能够产生多种单一基因的技术。然而，epPCR可以产生大量突变的基因变体，这对将这些突变的PCR产物连接到质粒载体中构成了挑战。通常，诱变PCR的引物包含与所选质粒相容的人工限制酶位点。

Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library’s breadth.

将产物切割并连接至线性化质粒，然后通过DNA连接酶再循环。然而，这种被称为连接依赖性过程克隆（LDCP）的剪切粘贴方法效率有限，因为潜在突变体的丢失不可避免地导致文库宽度的显着减少。

An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR.

LDCP的替代方法是循环聚合酶延伸克隆（CPEC）方法。该技术涉及一种反应，其中高保真DNA聚合酶延伸插入物和载体之间的重叠区域，形成环状分子。在这项研究中，我们的目的是比较传统的剪切粘贴酶法和CPEC从epPCR获得的编码红色荧光蛋白（DsRed2）的基因产生变异文库。

Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes..

我们的研究结果表明，与依赖限制性内切酶的方法相比，CPEC可以加速基因文库生成中的克隆过程，从而能够获得更多的基因变异。。

IntroductionProteins are biomolecules with significant potential for applications in research, the food and pharmaceutical industries1, and disease treatment2. Their amino acid sequences are determined by the information encoded in the genes. These sequences evolve due to spontaneous mutations, which are naturally selected by the environment or other factors.

引言蛋白质是一种生物分子，在研究，食品和制药工业1以及疾病治疗2中具有重要的应用潜力。它们的氨基酸序列由基因中编码的信息决定。这些序列是由于自发突变而进化的，自发突变是由环境或其他因素自然选择的。

Numerous technologies have been developed to study protein evolution in the laboratory. These aim to accelerate mutational rates, generate a vast array of variants, and facilitate the selection of proteins with desired characteristics. Different alterations to the polymerase chain reaction (PCR) method have made both site-specific and random mutagenesis more accessible.

已经开发了许多技术来研究实验室中的蛋白质进化。这些旨在加速突变率，产生大量变体，并促进选择具有所需特征的蛋白质。聚合酶链反应（PCR）方法的不同改变使得位点特异性和随机诱变更容易获得。

This has improved enzyme stability across broader pH and temperature ranges and increased tolerance to various organic solvents. There are many specific methods for protein engineering, which can be broadly categorized into two main groups: those based on the rational design of protein modifications and combinatorial methods that introduce changes randomly (For review see3).Random mutagenesis, for example, is a technology capable of generating high diversity from a single gene that involves the creation of libraries consisting of large numbers of genetic variants, where each member can be isolated and individually assessed concerning the genotype–phenotype relationship4.

这在更宽的pH和温度范围内提高了酶的稳定性，并提高了对各种有机溶剂的耐受性。蛋白质工程有许多特定的方法，可以大致分为两大类：基于蛋白质修饰的合理设计的方法和随机引入变化的组合方法（有关综述，请参见3）。例如，随机诱变是一种能够从单个基因产生高度多样性的技术，它涉及创建由大量遗传变异组成的文库，其中每个成员都可以被分离出来，并就基因型与表型的关系进行单独评估4。

Genetic variants can be obtained from methods such as error-prone PCR (epPCR). This last methodology, developed by Leung et al.5, uses a low-fidelity DNA polymerase that, under certain conditions, can introduce random mutations during PCR amplification of a target gene. The products of these mutagenic PCRs must then be linked to plasmid vectors and used to produce gene libraries.

遗传变异可以通过易错PCR（epPCR）等方法获得。Leung等人开发的最后一种方法使用了一种低保真DNA聚合酶，在某些条件下，该聚合酶可以在靶基因的PCR扩增过程中引入随机突变。然后必须将这些诱变PCR的产物与质粒载体连接并用于产生基因文库。

Al.

艾尔。

Data availability

数据可用性

The data and materials used in this study are available upon request from the corresponding author, D.V.A. The sequence data used in this study are available at https://www.ncbi.nlm.nih.gov for pCDF1b (Accession Number OR900361.1) and at https://www.uniprot.org for the plasmid pDsRed2 (Accession Number Q9U6Y8)..

https://www.ncbi.nlm.nih.gov对于pCDF1b（登录号OR900361.1）和https://www.uniprot.org对于质粒pDsRed2（登录号Q9U6Y8）。。

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Download referencesAcknowledgementsWe would like to express our gratitude to the Molecular Biology Laboratory at the Federal University of Rio Grande (FURG) for their assistance, resources, and expertise, which were instrumental in the successful completion of our research. This study was funded by National Council for Scientific and Technological Development (MCTI/CNPQ Call No.

下载参考文献致谢我们要感谢格兰德河联邦大学（FURG）分子生物学实验室的帮助，资源和专业知识，这些帮助我们成功完成了研究。这项研究由国家科学技术发展委员会（MCTI/CNPQ Call No.）资助。

454689/2014-4) and by Coordination for the Improvement of Higher Education Personnel (CAPES), which provided the master’s scholarship.Author informationAuthors and AffiliationsDepartamento de Biología, Universidad del Valle (UV), Cali, ColombiaNatalia Ossa-HernándezLaboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Rio Grande, BrazilLuis Fernando MarinsInstituto de Biologia, Universidade Federal de Pelotas - UFPEL, Campus Universitário Capão do Leão s/n, Pelotas, RS, 96160-000, BrazilDaniela Volcan AlmeidaAuthorsNatalia Ossa-HernándezView author publicationsYou can also search for this author in.

454689/2014-4）和提供硕士奖学金的高等教育人员改进协调会（CAPES）。作者信息作者和附属机构哥伦比亚卡利瓦莱大学生物系Natalia Ossa Hernández生物分子实验室Ciências Biológicas研究所、里约热内卢联邦大学（FURG）、巴西里约热内卢Luis Fernando MarinsInstituto de Biologia、佩洛塔联邦大学-UFPEL、佩洛塔斯大学校园Capão do Leãos/n，Pelotas，RS，96160-000、巴西Daniela Volcan Almeida作者Natalia OSa Herná作者出版物您也可以在中搜索此作者。

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PubMed Google ScholarContributionsAll authors (N.O.-H., L.F.M. and D.V.A.) were involved in the drafting and critical review of the manuscript and all contributed to the final writing. Experimental assays, results analysis, and writing were carried out by N.O.-H and D.V.A.Corresponding authorsCorrespondence to.

PubMed谷歌学术贡献所有作者（N.O.-H.，L.F.M.和D.V.A.）都参与了稿件的起草和批判性审查，并为最终写作做出了贡献。实验测定，结果分析和写作由N.O.-H和D.V.A进行。通讯作者通讯。

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Reprints and permissionsAbout this articleCite this articleOssa-Hernández, N., Marins, L.F. & Almeida, D.V. Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries.

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KeywordsDNA recombinationGene amplificationMolecular cloningRandom mutationsVariant genes

关键词DNA重组基因扩增分子克隆随机突变变异基因

Subjects

Expression systemsSynthetic biology

表达系统合成生物学

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