Nucleic Acids Research, 1985, Vol. 13, No. 24 8765-8785
© 1985
Articles |
The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie Hermann-Rein-Strasse 3, D-3400 Göttingen, FRG
Received October 7, 1985. Revised November 26, 1985. Accepted November 26, 1985.
M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotldic 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 M3mp2 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.
1Present address: Laboratory of Bioorgsnic Chemistry and Biochemistry, The Rockefeller University, 1234) York Avenue, New York, NY 10021-6399, USA
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|
|
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|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
K Basler, D Yen, A Tomlinson, and E Hafen Reprogramming cell fate in the developing Drosophila retina: transformation of R7 cells by ectopic expression of rough. Genes & Dev., May 1, 1990; 4(5): 728 - 739. [Abstract] [PDF]
|
|
|
|
|
|
S J Bray, B Burke, N H Brown, and J Hirsh Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element. Genes & Dev., August 1, 1989; 3(8): 1130 - 1145. [Abstract] [PDF]
|
|
|
|
|
|
W A Johnson, C A McCormick, S J Bray, and J Hirsh A neuron-specific enhancer of the Drosophila dopa decarboxylase gene. Genes & Dev., May 1, 1989; 3(5): 676 - 686. [Abstract] [PDF]
|
|
|
|
|
|
S. Tapscott, R. Davis, M. Thayer, P. Cheng, H Weintraub, and A. Lassar MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts Science, October 21, 1988; 242(4877): 405 - 411. [Abstract] [PDF]
|
|
|
|
|
|
K K Nelson and M R Green Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes & Dev., March 1, 1988; 2(3): 319 - 329. [Abstract] [PDF]
|
|
|
|
|
|
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|
|