A rapid and efficient method for site-directed mutagenesis using one- step overlap extension PCR

Urban, Andreas; Neukirchen, Susanne; Jaeger, Karl‐Erich

doi:10.1093/nar/25.11.2227

Cited by 184 publications

(140 citation statements)

References 9 publications

(6 reference statements)

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“…They were optimized for E. coli and reintroduced through one of the PCR primers. Mutations were constructed by site-directed mutagenesis with synthetic oligonucleotides and using previously published methods (Kunkel et al, 1987;Urban et al, 1997). All genetic constructs were verified by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Lisova

Hardy

Petit

et al. 2007

Journal of General Virology

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Dengue is caused by a taxonomic group of four viruses, dengue virus types 1-4 (DENV1-DENV4). A molecular understanding of the antibody-mediated protection against this disease is critical to design safe vaccines and therapeutics. Here, the energetic epitope of antibody mAb4E11, which neutralizes the four serotypes of DENV but no other flavivirus, and binds domain 3 (ED3) of their envelope glycoprotein, was characterized. Alanine-scanning mutagenesis of the ED3 domain from serotype DENV1 was performed and the affinities between the mutant domains and the Fab fragment of mAb4E11 were measured. The epitope residues (307-312, 387, 389 and 391) were at the edges of two distinct b-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency. INTRODUCTIONDengue is a re-emerging viral disease. It is caused by four types of virus, dengue virus types 1-4 (DENV1-DENV4), which belong to the species Dengue virus in the genus Flavivirus (Heinz et al., 2000). They are transmitted by Aedes mosquitoes and infect between 50 million and 100 million persons each year. The disease generally takes a mild form, dengue fever, but severe forms, dengue haemorragic fever and dengue shock syndrome, have recently become epidemic (Gubler, 2002).The immune response against an infection by DENV involves both a humoral component, in the form of neutralizing antibodies, and a cell-mediated component (Guzman & Kouri, 2002). The preferential reactivation of the memory B cells that correspond to a primary infection, and an antibody-dependent enhancement of infection, might constitute triggering mechanisms of the severe forms during a secondary infection by a different viral serotype (Halstead, 2003; Mongkolsapaya et al., 2003). A molecular understanding of the events that lead to antibody neutralization, enhancement or escape is critical to the development of efficient and secure vaccines and therapeutics. DENV1-DENV4 are enveloped RNA viruses, like all flaviviruses. The structures of the whole virus and of the envelope glycoprotein E (gpE) have been solved by electron cryomicroscopy and X-ray crystallography (Modis et al., 2003(Modis et al., , 2005 Zhang et al., 2003). Ninety dimers of gpE cover the surface of the virus. Each monomer comprises three ectodomains, ED1-ED3, and a transmembrane segment. ED2 includes the dimerization interface, a glycosylation site and the peptide of fusion with the cellular membrane. ED3 is continuou...

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Section: Methodsmentioning

confidence: 99%

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Lisova

Hardy

Petit

et al. 2007

Journal of General Virology

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“…Site-directed mutagenesis was done according to the one-step overlap extension PCR method (40). The PCR amplifications were performed with 35 cycles of denaturation (92°C, 30 s), annealing (55°C, 30 s), and extension (72°C, 1 min).…”

Section: Methodsmentioning

confidence: 99%

Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II

Chávez‐Gutiérrez¹,

Matta‐Camacho²,

Osuna³

et al. 2006

Journal of Biological Chemistry

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“…The resulting vector was digested with StuI, releasing the XP2RO cDNA sequence Ϫ62 to ϩ1317, which was inserted into pBacPAK8 (CLONTECH) at the StuI site to produce the transfection vector 2RO-p48/pBacPAK8. To construct the mutant XP82TO DDB2 transfection vector, the A 3 G mutation at nucleotide ϩ730 was introduced into the wild type DDB2 cDNA sequence by overlap extension PCR (21). Then the mutant DDB2 cDNA, containing nucleotides Ϫ11 to ϩ1317, was inserted into pBacPAK8 at the BamHI and StuI sites to produce the transfection vector 82TO-p48/pBacPAK8.…”

Section: Methodsmentioning

confidence: 99%

Human Damage-specific DNA-binding Protein p48

Nichols

Itoh

Graham

et al. 2000

Journal of Biological Chemistry

126

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Damage-specific DNA binding (DDB) activity purifies from HeLa cells as a heterodimer (p127 and p48) and is absent from cells of a subset (Ddb ؊ ) of xeroderma pigmentosum Group E (XPE) patients. Each subunit was overexpressed in insect cells and purified. Both must be present for the damaged DNA band shift characteristic of the HeLa heterodimer. However, overexpressed p48 peptides containing the mutations found in three Ddb ؊ XPE strains are inactive, and wild type p48 restores DDB activity to extracts from a fourth XPE Ddb ؊ strain, GM01389, in which compound heterozygous mutations in DDB2 (p48) lead to a L350P change from one allele and a Asn-349 deletion from the other. Although these results indicate that these mutations are each responsible for the loss of DDB activity, they do not affect nuclear localization of p48. In normal fibroblasts, a 4-fold increase in p48 mRNA amount was observed 38 h after UV irradiation, preceding a similar elevation in p48 protein and DDB activity at 48 h, implying that p48 limits DDB activity in vivo. Because DNA repair is virtually complete before 48 h, a role for DDB other than DNA repair is suggested.The rare human hereditary disease, xeroderma pigmentosum (XP), 1 is characterized biochemically by defective nucleotide excision repair (NER), which manifests clinically as sensitivity to ultraviolet light and a high incidence of skin cancer. Based on fusion studies of cells from XP patients, seven NERdefective complementation groups (A through G) and a postreplication repair-deficient variant group (XPV) have been identified (1, 2). Cell strains from a subset (Ddb Ϫ ) of individuals carrying XP complementation group E (XPE) lack a damage-specific DNA binding (DDB) activity (3-5). Because DDB was reported to recognize many types of DNA lesions (6 -11) and is inducible by treatment with DNA-damaging agents (7, 12, 13), DDB was originally expected to play a role in damage recognition prior to nucleotide excision repair. However, recent NER reconstitution studies have reported that DDB is not required in vitro (14 -16). Nonetheless, microinjection of purified HeLa DDB heterodimer (p127, p48) into XPE cells restores in vivo DNA repair synthesis to normal levels in XPE Ddb Ϫ strains but not in XPE Ddb ϩ strains or in cells from other XP groups (17). Sequencing of the cDNAs that encode the DDB heterodimer have identified single base mutations only in DDB2 (p48) of XPE Ddb Ϫ cells. In the Ddb Ϫ strains, XP2RO and XP3RO, a G 3 A transition at nucleotide ϩ818 causes an R273H change in p48, whereas an A 3 G transition at nucleotide ϩ730 causes a K244E change in XP82TO. Overexpression of wild type p48, but not of p127, in insect cells greatly increases DDB activity in extracts prepared from these cells, indicating that p48 is required for damage-specific DNA binding (18).In the present study, we have reconstituted human DDB activity in an electrophoretic mobility shift assay by combining insect extracts containing individually overexpressed wild type p127 and p48. Both extracts were requir...

show abstract

A rapid and efficient method for site-directed mutagenesis using one- step overlap extension PCR

Cited by 184 publications

References 9 publications

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II

Human Damage-specific DNA-binding Protein p48

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