{"title":"内部提取和纯化高处理能力 DNA 聚合酶 Pfu-Sso7d","authors":"Aisha Mahboob, Nishat Fatma, Afzal Husain","doi":"10.21769/BioProtoc.4967","DOIUrl":null,"url":null,"abstract":"The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase\",\"authors\":\"Aisha Mahboob, Nishat Fatma, Afzal Husain\",\"doi\":\"10.21769/BioProtoc.4967\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.\",\"PeriodicalId\":93907,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.0000,\"publicationDate\":\"2024-04-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"0\",\"ListUrlMain\":\"https://doi.org/10.21769/BioProtoc.4967\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"0","ListUrlMain":"https://doi.org/10.21769/BioProtoc.4967","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
聚合酶链反应(PCR)是一种广泛使用的技术,可快速准确地复制多个特定 DNA 片段。除了天然存在的 DNA 聚合酶外,PCR 还利用一系列经过基因修饰的重组 DNA 聚合酶,每种聚合酶都具有不同程度的处理能力和保真度。Pfu-Sso7d 是一种融合 DNA 聚合酶,由 Sso7d(一种小型 DNA 结合蛋白)与 Pfu DNA 聚合酶融合而成。Pfu-Sso7d 以其高加工性、高效率和高保真度而著称,但以各种商品名和商业变体出售,价格高得吓人。我们最近报道了一种快速简便的纯化方案,利用乙醇或丙酮从热清除裂解液中沉淀 Pfu-Sso7d。我们还优化了一种 PCR 缓冲溶液,当与 Pfu-Sso7d 一起使用时,其效果优于商用缓冲液。在此,我们将逐步介绍如何纯化重组 Pfu-Sso7d。该纯化方案和缓冲液系统将为研究人员提供具有成本效益的融合聚合酶。主要特点 - 我们详细介绍了利用乙醇和丙酮纯化 Pfu-Sso7d 的沉淀方案。- 尽管乙醇和丙酮都能有效提高沉淀效率,但丙酮因其卓越的性能而更受青睐。- 此外,我们还介绍了一种性能优于市售 PCR 缓冲液的 PCR 缓冲液。- 内部纯化的 Pfu-Sso7d 和所述 PCR 缓冲液在 PCR 应用中表现出卓越的性能。
In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase
The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.