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The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA
Open accessAbstract
M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the (-)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.
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Cited by
Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J., Hudson, P., & Winter, G. (1991) · Nucleic Acids Research · doi:10.1093/nar/19.15.4133 · Cited by 1,020
Gutierrez, L., Magee, A. I., Marshall, C. J., & Hancock, J. F. (1989) · The EMBO Journal · doi:10.1002/j.1460-2075.1989.tb03478.x · Cited by 460
Malim, M. H., Tiley, L. S., McCarn, D. F., Rusche, J. R., Hauber, J., & Cullen, B. R. (1990) · Cell · doi:10.1016/0092-8674(90)90670-a · Cited by 430
Janzon, L., & Arvidson, S. (1990) · The EMBO Journal · doi:10.1002/j.1460-2075.1990.tb08254.x · Cited by 296
Carter, P. (1986) · Biochemical Journal · doi:10.1042/bj2370001 · Cited by 289
Metadata
- Authors
- Taylor, W. J., Ott, J., & Eckstein, F.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 1985
- Volume
- 13
- Issue
- 24
- Pages
- 8765-8785
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2042869569
- Cited by
- 833
- References
- 0
- FWCI
- 10.50
- Type
- journal article
- Language
- en
- Open access
- green
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- Taylor, W. J., Ott, J., & Eckstein, F. (1985). The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Research. https://doi.org/10.1093/nar/13.24.8765
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