A Universal Nested Deletion Method Using an Arbitrary Primer and Elimination of a Unique Restriction Site

Zhu, Li; Holtz, Ann E.

doi:10.1385/0-89603-332-5:119

Cited by 2 publications

(1 citation statement)

References 2 publications

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“…Once the methodology and its effectiveness have been established, other possible future work will be to change the size and number of blocks to be deleted or inserted in addition to changing the location where such mutations are introduced. This will be equivalent to a high‐throughput version of the previous efforts performed on nested deletion [17,18] or shuffling of proteins such as hemoglobin [19]. These works are useful not only from a molecular engineering viewpoint (such as creating minimum‐sized GFP) but also from a protein science viewpoint (such as exploring the protein sequence space).…”

Section: Resultsmentioning

confidence: 99%

GFPs of insertion mutation generated by molecular size‐altering block shuffling

2003

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Insertion and deletion analyses of a protein have been less common than point mutation analyses, partly due to the lack in e¡ective methods. This is the case with the green £uo-rescent protein (GFP), which is so widely applied in molecular biology and other ¢elds. In this paper we ¢rst introduce a systematic approach for generating insertion/deletion mutants of GFP. A new technology of Y-ligation-based block shu¥ing (YLBS) was successfully applied to produce size-altered GFPs, providing insertion-containing GFPs of £uorescence, though no deletion type of £uorescence was obtained so far as examined. The analysis of these proteins suggested that size alteration (deletion/insertion) is acceptable so far as some type of rearrangement in a local structure can accommodate it. This paper demonstrates that YLBS can generate insertion and deletion mutant libraries systematically, which are bene¢cial in the study of structure^function relationship. ß

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Section: Resultsmentioning

confidence: 99%

GFPs of insertion mutation generated by molecular size‐altering block shuffling

2003

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A Novel Simple and Rapid PCR-Based Site-Directed Mutagenesis Method

Rabhi

Guedel

Chouk

et al. 2004

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Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5' end of one of the primers, whereas the other primer starts with the nucleotide at position (-1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat alpha integrin CD11b A-domain and the human CD8beta cloned in pPICZalphaB, pGEX-2T, and CDM8 expression vectors, respectively.

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A Universal Nested Deletion Method Using an Arbitrary Primer and Elimination of a Unique Restriction Site

Cited by 2 publications

References 2 publications

GFPs of insertion mutation generated by molecular size‐altering block shuffling

GFPs of insertion mutation generated by molecular size‐altering block shuffling

A Novel Simple and Rapid PCR-Based Site-Directed Mutagenesis Method

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