在大肠杆菌中对PCR产物进行体内克隆。
In vivo cloning of PCR products in E. coli.
作者信息
Oliner J D, Kinzler K W, Vogelstein B
机构信息
Johns Hopkins Oncology Center, Baltimore, MD 21231.
出版信息
Nucleic Acids Res. 1993 Nov 11;21(22):5192-7. doi: 10.1093/nar/21.22.5192.
Abstract
This report describes an efficient method to clone PCR products exploiting endogenous Escherichia coli enzymatic activities. PCR products are engineered to contain terminal sequences identical to sequences at the two ends of a linearized vector. PCR products and vector DNA are then simply co-transfected into E. coli strain JC8679, obviating the requirement for enzymatic treatment of the PCR product or in vitro ligation. The high rate of homologous recombination in this strain results in efficient incorporation of the insert into the vector, a process we refer to as in vivo cloning (IVC).
摘要
本报告描述了一种利用大肠杆菌内源性酶活性克隆PCR产物的有效方法。对PCR产物进行工程改造，使其包含与线性化载体两端序列相同的末端序列。然后将PCR产物和载体DNA简单地共转染到大肠杆菌菌株JC8679中，无需对PCR产物进行酶处理或体外连接。该菌株中同源重组的高发生率导致插入片段高效整合到载体中，我们将这一过程称为体内克隆（IVC）。
相似文献
1
In vivo cloning of PCR products in E. coli.在大肠杆菌中对PCR产物进行体内克隆。
Nucleic Acids Res. 1993 Nov 11;21(22):5192-7. doi: 10.1093/nar/21.22.5192.
2
Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.通过大肠杆菌中的同源重组实现PCR片段的最佳克隆
PLoS One. 2015 Mar 16;10(3):e0119221. doi: 10.1371/journal.pone.0119221. eCollection 2015.
3
High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.利用大肠杆菌中的同源重组，高通量克隆人肝脏完整开放阅读框。
Anal Biochem. 2010 Feb 15;397(2):162-7. doi: 10.1016/j.ab.2009.10.018. Epub 2009 Oct 14.
4
Restrictionless cloning.无限制克隆
Methods Enzymol. 2013;529:125-34. doi: 10.1016/B978-0-12-418687-3.00009-4.
5
FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.FastCloning：一种高度简化、无需纯化、无需序列和连接的 PCR 克隆方法。
BMC Biotechnol. 2011 Oct 12;11:92. doi: 10.1186/1472-6750-11-92.
6
Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.无限制酶切连接（RLF）克隆：一种通过PCR产物的体内同源重组实现的高通量克隆方法。
Genet Mol Res. 2015 Oct 9;14(4):12306-15. doi: 10.4238/2015.October.9.19.
7
Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors.基于酵母的多DNA片段高效体内DNA克隆及酵母/大肠杆菌穿梭载体的同步构建。
Biotechniques. 2006 Jan;40(1):79-83. doi: 10.2144/000112041.
8
Directional cloning of blunt-ended PCR products.平端PCR产物的定向克隆
Biotechniques. 1993 Sep;15(3):502-5.
9
An efficient method for blunt-end ligation of PCR products.一种用于PCR产物平端连接的高效方法。
Biotechniques. 1992 Jan;12(1):28, 30.
10
A vector for facile PCR product cloning and modification generating any desired 4-base 5' overhang: pRPM.一种用于便捷PCR产物克隆和修饰以产生任何所需4碱基5'突出端的载体：pRPM。
Biotechniques. 1993 Feb;14(2):250-5.
引用本文的文献
1
The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium sp. Tol 5.消除两个限制酶基因可实现非感受态革兰氏阴性菌 Tol 5 的电穿孔转化和基于 CRISPR-Cas9 的碱基编辑。
Appl Environ Microbiol. 2024 Jun 18;90(6):e0040024. doi: 10.1128/aem.00400-24. Epub 2024 May 9.
2
Alternative PCR-Based Approaches for Generation of Strains.基于聚合酶链式反应的菌株构建替代方法。
Microorganisms. 2023 Sep 12;11(9):2297. doi: 10.3390/microorganisms11092297.
3
PNPLA3(I148M) Inhibits Lipolysis by Perilipin-5-Dependent Competition with ATGL.载脂蛋白 L3（I148M）通过与脂肪甘油三酯酶竞争结合 perilipin-5 抑制脂肪分解。
Cells. 2022 Dec 24;12(1):73. doi: 10.3390/cells12010073.
4
Past, Present, and Future of Genome Modification in .……中基因组编辑的过去、现在与未来 （原句不完整，翻译可能存在不准确之处）
Microorganisms. 2022 Sep 14;10(9):1835. doi: 10.3390/microorganisms10091835.
5
Reduction of Contributes to a Protection Against Atherosclerosis.降低……有助于预防动脉粥样硬化。
你提供的原文似乎不完整，“Reduction of ”后面缺少具体内容。
Front Cardiovasc Med. 2022 Mar 11;9:818662. doi: 10.3389/fcvm.2022.818662. eCollection 2022.
6
Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters.天然产物生物合成基因簇克隆策略的最新进展
Front Bioeng Biotechnol. 2021 Jul 13;9:692797. doi: 10.3389/fbioe.2021.692797. eCollection 2021.
7
RecA-independent recombination: Dependence on the Escherichia coli RarA protein.RecA 独立重组：依赖于大肠杆菌 RarA 蛋白。
Mol Microbiol. 2021 Jun;115(6):1122-1137. doi: 10.1111/mmi.14655. Epub 2020 Dec 19.
8
Cloning, expression and purification of the low-complexity region of RanBP9 protein.RanBP9 蛋白低复杂度区域的克隆、表达与纯化。
Protein Expr Purif. 2020 Aug;172:105630. doi: 10.1016/j.pep.2020.105630. Epub 2020 Mar 23.
9
DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning.利用常见的实验室细菌进行 DNA 组装：一种简化分子克隆的新兴工具。
J Biol Chem. 2019 Oct 18;294(42):15271-15281. doi: 10.1074/jbc.REV119.009109. Epub 2019 Sep 14.
10
Manipulating the Mouse Genome Using Recombineering.利用重组工程技术操控小鼠基因组
Adv Genet Eng. 2013;2(2). doi: 10.4172/2169-0111.1000108. Epub 2013 Jun 27.
本文引用的文献
1
Rapid cloning by homologous recombination in vivo.体内同源重组快速克隆
Nucleic Acids Res. 1993 Jul 25;21(15):3601-2. doi: 10.1093/nar/21.15.3601.
2
Direct cloning and sequence analysis of enzymatically amplified genomic sequences.酶促扩增基因组序列的直接克隆与序列分析
Science. 1986 Sep 5;233(4768):1076-8. doi: 10.1126/science.3461561.
3
Double-stranded gap repair of DNA by gene conversion in Escherichia coli.大肠杆菌中通过基因转换进行的DNA双链缺口修复
Genetics. 1988 Aug;119(4):751-7. doi: 10.1093/genetics/119.4.751.
4
Whole genome PCR: application to the identification of sequences bound by gene regulatory proteins.全基因组PCR：在鉴定基因调控蛋白结合序列中的应用。
Nucleic Acids Res. 1989 May 25;17(10):3645-53. doi: 10.1093/nar/17.10.3645.
5
A simple method for site-directed mutagenesis using the polymerase chain reaction.一种利用聚合酶链反应进行定点诱变的简单方法。
Nucleic Acids Res. 1989 Aug 25;17(16):6545-51. doi: 10.1093/nar/17.16.6545.
6
Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases.原核生物和真核生物DNA聚合酶催化的新型非模板核苷酸添加反应。
Nucleic Acids Res. 1988 Oct 25;16(20):9677-86. doi: 10.1093/nar/16.20.9677.
7
Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column.通过一次性塑料柱离心从琼脂糖凝胶中快速纯化DNA。
Anal Biochem. 1987 Jan;160(1):115-8. doi: 10.1016/0003-2697(87)90620-8.
8
Ligation-independent cloning of PCR products (LIC-PCR).聚合酶链式反应（PCR）产物的不依赖连接酶克隆（LIC-PCR）。
Nucleic Acids Res. 1990 Oct 25;18(20):6069-74. doi: 10.1093/nar/18.20.6069.
9
Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites.高效克隆含末端限制性内切酶识别位点的PCR扩增DNA。
Nucleic Acids Res. 1990 Oct 25;18(20):6156. doi: 10.1093/nar/18.20.6156.
10
Restriction endonuclease cleavage at the termini of PCR products.在PCR产物末端进行限制性内切核酸酶切割。
Biotechniques. 1990 Sep;9(3):304, 306.
Zotero 插件