関: insertional activation、insertional mutagenesis

WordNet

a container that holds a magnetic tape used for recording or playing sound or video
an event capable of causing a mutation

PrepTutorEJDIC

(録音・録画テープの)『カセット』;(タイプライターの)カートリッジ,(フイルムの)パトローネ

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/02/03 18:39:55」(JST)

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Cassette mutagenesis is a type of site-directed mutagenesis that uses a short, double-stranded oligonucleotide sequence (gene cassette) to replace a fragment of target DNA. It uses complimentary restriction enzyme digest ends on the target DNA and gene cassette to achieve specificity. It is different from methods that use single oligonucleotide in that a single gene cassette can contain multiple mutations. Unlike many site directed mutagenesis methods, cassette mutagenesis also does not involve primer extension by DNA polymerase.^[1]^[2]^[3]^[4]^[5]

Mechanism

First, restriction enzymes are used to cleave near the target sequence on DNA contained in a suitable vector. This step removes the target sequence and everything between the restriction sites. Then, the synthetic double stranded DNA containing the desired mutation and ends that are complimentary to the restriction digest ends are ligated in place of the sequence removed. Finally, the resultant construct is sequenced to check that the target sequence contains the intended mutation.^[1]

Usage

The use of synthetic gene cassette allows total control over the type of mutation that can be generated. When studying protein functions, cassette mutagenesis can allow a scientist to change individual amino acids by introducing different codons or omitting codons. ^[1]^[2]

By including the SD sequence and the first few codons of a gene, a scientist can easily and dramatically affect the expression level of a protein by altering these regulatory sequences.^[2]

Limitations

To use this method, the sequence of the target sequence and nearby restriction sites must be known. Since restriction enzymes are used, for this method to be useful, the restriction sites flanking the target DNA has to be unique in the gene/vector system so that the gene cassette can be inserted with specificity. The length of the sequence flanked by the restriction sites is also a limiting factor due to the use of synthetic gene cassettes.^[2]^[3]

Advantages

Since one gene cassette can contain multiple mutations, less total oligonucleotide synthesis and purification is needed. Compared to mutagenesis methods that requires the synthesis of double stranded DNA using a single stranded template (1-30% in vitro in M13), the efficiency of the ligation of oligodeoxynucleotide cassette is close to 100%. The high efficiency of the mutagenesis means mutants can be screened directly by sequencing.^[2] Once the vector is set up with flanking restriction sites, all manipulations (i.e., mutagenesis, sequencing, expression) can be performed in the same plasmid.^[2]

References

^ ^a ^b ^c Worrall, Andrew (1994). "Site-Directed Mutagenesis by the Cassette Method". Methods in Molecular Biology 30. Humana Press. pp. 199–210. ISBN 978-1-59259-517-4. PMID 8004195. Retrieved Nov 26, 2014.
^ ^a ^b ^c ^d ^e ^f Wells, J. A.; Vasser, M; Powers, D. B. (1985). "Cassette mutagenesis: An efficient method for generation of multiple mutations at defined sites". Gene 34 (2-3): 315–23. PMID 3891521. edit
^ ^a ^b Clore, Adam; Reinertson, Brian; Rose, Scott; Sabel, Jaime. "Ultramer Oligonucleotides Mutagenesis Application Guide - Experimental Overview, Protocol, Troubleshooting". WWW.IDTDNA.COM. Intergrated DNA Technologies. p. 16. Retrieved Nov 6, 2014.
^ Kegler-Ebo, D. M.; Docktor, C. M.; Dimaio, D (1994). "Codon cassette mutagenesis: A general method to insert or replace individual codons by using universal mutagenic cassettes". Nucleic acids research 22 (9): 1593–9. PMC 308034. PMID 8202358. edit
^ El-Mansi, E. M. T.; Bryce, C. F. A.; Demain, Arnold L.; A.R. Allman (2006-10-25). Fermentation Microbiology and Biotechnology, Second Edition. CRC Press. pp. 222–. ISBN 9780849353345. Retrieved 27 November 2014. Cite uses deprecated parameters (help)

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English Journal

Cytological characterization of murine bone marrow and spleen hematopoietic compartments for improved assessment of toxicity in preclinical gene marking models.

Yang M, Büsche G, Ganser A, Li Z.SourceInstitute of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.
Annals of hematology.Ann Hematol.2013 May;92(5):595-604. doi: 10.1007/s00277-012-1655-3. Epub 2013 Jan 10.
Gene therapy has proven its potential to cure diseases of the hematopoietic system, but potential adverse reactions related to insertional mutagenesis by integrating gene vectors and chromosomal instability in long-lived repopulating cells have emerged as a major limitation. Preclinical gene therapy
PMID 23307598

Protein contacts and ligand binding in the inward-facing model of human p-glycoprotein.

Pajeva IK, Hanl M, Wiese M.SourceInstitute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 105, 1113 Sofia (Bulgaria). pajeva@biomed.bas.bg.
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The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward-open homology model of human P-glycoprotein (P-gp). The analysis revealed communication nets thr
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Genome-wide association study of sleep in Drosophila melanogaster.

Harbison ST, McCoy LJ, Mackay TF.AbstractBACKGROUND: Sleep is a highly conserved behavior, yet its duration and pattern vary extensively among species and between individuals within species. The genetic basis of natural variation in sleep remains unknown.
BMC genomics.BMC Genomics.2013 Apr 25;14(1):281. [Epub ahead of print]
BACKGROUND: Sleep is a highly conserved behavior, yet its duration and pattern vary extensively among species and between individuals within species. The genetic basis of natural variation in sleep remains unknown.RESULTS: We used the Drosophila Genetic Reference Panel (DGRP) to perform a genome-wid
PMID 23617951

Japanese Journal

トランスポゾンに基づく持続発現型ベクターの開発と治療応用

中西秀之,樋口ゆり子,川上茂 [他],山下富義,橋田充
YAKUGAKU ZASSHI 129(12), 1433-1443, 2009
… One is the transposon containing transgenes, and the other is the expression cassette of the transposase. … Although transposon-based integrative vector systems have problems, such as insertional mutagenesis, studies to overcome these problems have been progressing, and these vector systems will become indispensable tools to cure refractory diseases. …
NAID 130000136134

Quinoprotein Alcohol Dehydrogenase Is Involved in Catabolic Acetate Production, while NAD-Dependent Alcohol Dehydrogenase in Ethanol Assimilation in Acetobacter pasteurianus SKU1108(MICROBIAL PHYSIOLOGY AND BIOTECHNOLOGY)

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Journal of bioscience and bioengineering 96(6), 564-571, 2003-12-25
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NAID 110002665243

Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis

HE Y. A.
Biochemistry 36, 8831-8839, 1997
NAID 80009812698

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リンク元	「カセット変異導入法」「insertional activation」「insertional mutagenesis」「カセット変異導入」
関連記事	「mutagenesis」