利用氨苄青霉素选择进行高效的定点体外诱变。
Efficient site directed in vitro mutagenesis using ampicillin selection.
作者信息
Lewis M K, Thompson D V
机构信息
Promega Corporation, Madison, WI 53711.
出版信息
Nucleic Acids Res. 1990 Jun 25;18(12):3439-43. doi: 10.1093/nar/18.12.3439.
Abstract
A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.
摘要
描述了一种新型质粒载体pSELECT-1，其可用于高效的体外定点诱变。诱变方法基于使用单链DNA和两种引物，一种诱变引物和第二种校正引物，后者校正载体上氨苄青霉素抗性基因中的缺陷并使载体恢复氨苄青霉素抗性。使用T4 DNA聚合酶和T4 DNA连接酶，两种引物在模板上物理连接。通过在氨苄青霉素存在下生长来选择非突变DNA链。在载体测试中，获得了高效（60-90%）的诱变效果。
相似文献
1
Efficient site directed in vitro mutagenesis using ampicillin selection.利用氨苄青霉素选择进行高效的定点体外诱变。
Nucleic Acids Res. 1990 Jun 25;18(12):3439-43. doi: 10.1093/nar/18.12.3439.
2
Efficient site-directed mutagenesis by simultaneous use of two primers.通过同时使用两种引物进行高效的定点诱变。
Nucleic Acids Res. 1983 Aug 11;11(15):5103-12. doi: 10.1093/nar/11.15.5103.
3
An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis.通过寡核苷酸介导诱变构建的改良型阳性选择质粒载体。
Nucleic Acids Res. 1983 Nov 25;11(22):8019-30. doi: 10.1093/nar/11.22.8019.
4
Solid phase in vitro mutagenesis using plasmid DNA template.使用质粒DNA模板的固相体外诱变
Nucleic Acids Res. 1990 Sep 11;18(17):5107-12. doi: 10.1093/nar/18.17.5107.
5
Primer-directed mutagenesis of linearized plasmids.线性化质粒的引物定向诱变
Anal Biochem. 1984 Dec;143(2):341-9. doi: 10.1016/0003-2697(84)90673-0.
6
Subcloning with new ampicillin- and kanamycin-resistant analogs of pUC19.使用pUC19新的氨苄青霉素和卡那霉素抗性类似物进行亚克隆。
Biotechniques. 1991 Apr;10(4):469-70, 472.
7
Method for cloning single-stranded oligonucleotides in a plasmid vector.在质粒载体中克隆单链寡核苷酸的方法。
Biotechniques. 1989 Apr;7(4):356-9.
8
A marker-coupled method for site-directed mutagenesis.
Gene. 1991 Jul 15;103(1):73-7. doi: 10.1016/0378-1119(91)90393-p.
9
Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.使用一个引物和环状质粒DNA模板进行简单高效的寡核苷酸定向诱变。
Gene Anal Tech. 1989 Jul-Aug;6(4):67-70. doi: 10.1016/0735-0651(89)90017-4.
10
Site-directed mutagenesis using positive antibiotic selection.利用阳性抗生素筛选进行定点诱变。
Mol Biotechnol. 1997 Apr;7(2):181-8. doi: 10.1007/BF02761754.
引用本文的文献
1
Fine tuning of the DNAM-1/TIGIT/ligand axis in mucosal T cells and its dysregulation in pediatric inflammatory bowel diseases (IBD).黏膜 T 细胞中 DNAM-1/TIGIT/配体轴的精细调节及其在儿科炎症性肠病（IBD）中的失调。
Mucosal Immunol. 2019 Nov;12(6):1358-1369. doi: 10.1038/s41385-019-0208-7. Epub 2019 Oct 3.
2
Fnr-, NarP- and NarL-dependent regulation of transcription initiation from the Haemophilus influenzae Rd napF (periplasmic nitrate reductase) promoter in Escherichia coli K-12.大肠杆菌K-12中流感嗜血杆菌Rd napF（周质硝酸还原酶）启动子转录起始的Fnr、NarP和NarL依赖性调控
J Bacteriol. 2005 Oct;187(20):6928-35. doi: 10.1128/JB.187.20.6928-6935.2005.
3
Dual overlapping promoters control napF (periplasmic nitrate reductase) operon expression in Escherichia coli K-12.双重重叠启动子控制大肠杆菌K-12中napF（周质硝酸还原酶）操纵子的表达。
J Bacteriol. 2003 Oct;185(19):5862-70. doi: 10.1128/JB.185.19.5862-5870.2003.
4
Synthetic lac operator substitutions for studying the nitrate- and nitrite-responsive NarX-NarL and NarQ-NarP two-component regulatory systems of Escherichia coli K-12.用于研究大肠杆菌K-12中硝酸盐和亚硝酸盐响应性NarX-NarL和NarQ-NarP双组分调节系统的合成乳糖操纵子替代物。
J Bacteriol. 2003 Apr;185(7):2104-11. doi: 10.1128/JB.185.7.2104-2111.2003.
5
The use of site-directed mutagenesis, transient transfection, and radioligand binding. A method for the characterization of receptor-ligand interactions.定点诱变、瞬时转染和放射性配体结合的应用。一种表征受体-配体相互作用的方法。
Mol Biotechnol. 2000 Jan;14(1):25-45. doi: 10.1385/MB:14:1:25.
6
General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al.产酸克雷伯菌M5al中硝酸盐同化调节基因nasR表达的一般氮调控
J Bacteriol. 1999 Dec;181(23):7274-84. doi: 10.1128/JB.181.23.7274-7284.1999.
7
Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum.酵母蛋白二硫键异构酶中的活性位点突变会导致对二硫苏糖醇敏感，并降低内质网中蛋白质折叠的速率。
J Cell Biol. 1997 Sep 22;138(6):1229-38. doi: 10.1083/jcb.138.6.1229.
8
The global regulator CsrA of Escherichia coli is a specific mRNA-binding protein.大肠杆菌的全局调控因子CsrA是一种特异性mRNA结合蛋白。
J Bacteriol. 1997 Jul;179(14):4639-42. doi: 10.1128/jb.179.14.4639-4642.1997.
9
Site-directed mutagenesis using positive antibiotic selection.利用阳性抗生素筛选进行定点诱变。
Mol Biotechnol. 1997 Apr;7(2):181-8. doi: 10.1007/BF02761754.
10
Competition between folding and glycosylation in the endoplasmic reticulum.内质网中折叠与糖基化之间的竞争。
EMBO J. 1996 Jul 15;15(14):3538-46.
本文引用的文献
1
Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli.大肠杆菌的甲基化导向DNA错配修复系统对不同的碱基/碱基错配进行校正的效率不同。
Cell. 1984 Oct;38(3):879-87. doi: 10.1016/0092-8674(84)90283-6.
2
Revised sequence of the tetracycline-resistance gene of pBR322.pBR322四环素抗性基因的修订序列。
Gene. 1983 May-Jun;22(2-3):277-80. doi: 10.1016/0378-1119(83)90112-9.
3
Strand displacement during deoxyribonucleic acid synthesis at single strand breaks.单链断裂处脱氧核糖核酸合成过程中的链置换。
J Biol Chem. 1971 Apr 25;246(8):2692-701.
4
DNA synthesis on a double-stranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwinding protein.T4噬菌体DNA聚合酶和T4基因32解旋蛋白在双链DNA模板上进行DNA合成。
J Biol Chem. 1974 Sep 10;249(17):5668-76.
5
DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues.大肠杆菌中的DNA错配修复可对抗5-甲基胞嘧啶残基的水解脱氨作用。
EMBO J. 1987 Jun;6(6):1809-15. doi: 10.1002/j.1460-2075.1987.tb02435.x.
6
In vitro mutagenesis.体外诱变
Annu Rev Genet. 1985;19:423-62. doi: 10.1146/annurev.ge.19.120185.002231.
7
A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants.
Gene. 1988 May 15;65(1):129-33. doi: 10.1016/0378-1119(88)90425-8.
8
Mutagenesis at a specific position in a DNA sequence.DNA序列中特定位置的诱变。
J Biol Chem. 1978 Sep 25;253(18):6551-60.
9
Complete nucleotide sequence of the Escherichia coli plasmid pBR322.大肠杆菌质粒pBR322的完整核苷酸序列。
Cold Spring Harb Symp Quant Biol. 1979;43 Pt 1:77-90. doi: 10.1101/sqb.1979.043.01.013.
Zotero 插件