{"title":"利用含脱氧肌苷寡核苷酸和核酸内切酶V从含有随机区域的片段体外构建大规模DNA文库","authors":"Yasuhide Yamamoto, Takuya Terai, Shigefumi Kumachi, Naoto Nemoto*","doi":"10.1021/acscombsci.9b00167","DOIUrl":null,"url":null,"abstract":"<p >Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 10<sup>13</sup> species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5′ end. Treatment of the PCR products with endonuclease V generates 3′ overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.</p>","PeriodicalId":14,"journal":{"name":"ACS Combinatorial Science","volume":"22 4","pages":"165–171"},"PeriodicalIF":3.7840,"publicationDate":"2020-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1021/acscombsci.9b00167","citationCount":"2","resultStr":"{\"title\":\"In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V\",\"authors\":\"Yasuhide Yamamoto, Takuya Terai, Shigefumi Kumachi, Naoto Nemoto*\",\"doi\":\"10.1021/acscombsci.9b00167\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 10<sup>13</sup> species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5′ end. Treatment of the PCR products with endonuclease V generates 3′ overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.</p>\",\"PeriodicalId\":14,\"journal\":{\"name\":\"ACS Combinatorial Science\",\"volume\":\"22 4\",\"pages\":\"165–171\"},\"PeriodicalIF\":3.7840,\"publicationDate\":\"2020-03-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1021/acscombsci.9b00167\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Combinatorial Science\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acscombsci.9b00167\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Chemistry\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Combinatorial Science","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acscombsci.9b00167","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Chemistry","Score":null,"Total":0}
In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V
Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5′ end. Treatment of the PCR products with endonuclease V generates 3′ overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.
期刊介绍:
The Journal of Combinatorial Chemistry has been relaunched as ACS Combinatorial Science under the leadership of new Editor-in-Chief M.G. Finn of The Scripps Research Institute. The journal features an expanded scope and will build upon the legacy of the Journal of Combinatorial Chemistry, a highly cited leader in the field.