Mutagenesis methods and compositions

Details Mutagenesis methods and compositions Mutagenesis methods and compositions

1993-12-28

1997-12-30

Degen, Nancy

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

435 911, 435 914, 435 9141, 435 9142, 536 2433, 536 253, C12N 1501, C12N 1511, C12N 1563, C07H 2104

Patent

active

057029318

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates generally to the oligonucleotide-directed mutagenesis of nucleic acids.
Oligonucleotide-directed mutagenesis is a valuable tool for the study of DNA function and protein structure and function, having become the method of choice to introduce predetermined structural changes into DNA. Such manipulation permits the alteration of a DNA sequence in order to determine its function, or may permit the production of reagents with commercial or medical significance. A number of different methods have been reported. See, inter alia, M. Smith, Ann. Rev. Genet. (1985) 19:423 for a review; and Section IV, Chapters 17-21, Meth. Enzymol. (1987) 154:329-414, both incorporated herein by reference.
Oligonucleotide-directed mutagenesis is accomplished by annealing to single-stranded DNA (ssDNA) a synthetic oligonucleotide which is complementary to the single-stranded template, except for an internal mismatch which directs the required change (point mutation, multiple mutation, insertion or deletion), resulting in the formation of a mutant-wild-type heteroduplex. There have been several adaptations of this basic concept. In most methods, following hybridization with the single-stranded target DNA, the oligonucleotide is extended with DNA polymerase to create a double-stranded structure. It is possible, in a "gapped duplex" approach, to forego the extension of the oligonucleotide with polymerase (W. Kramer and H.-J. Fritz, Meth. Enzymol. 154:350 (1987), incorporated herein by reference). In either case, the double-stranded DNA is then transformed into an E. coli host.
Theoretically, the yield of mutants using this procedure should be 50% due to the semi-conservative mode of DNA replication. That is, as cells transformed with the double-stranded heteroduplex DNA replicate each of the strands, one-half of their progeny will theoretically contain either wild-type or mutated DNA. In practice, however, the mutant yield may be much lower, often only a few percent or less. This is assumed to be due to such factors as incomplete in vitro polymerization, primer displacement by the DNA polymerase used in the fill-in reaction, and in vivo host-directed mismatch repair mechanisms which favor repair of the unmethylated newly synthesized DNA strand (B. Kramer et al., Cell 38:879 (1984)). Several methods have been described which increase the likelihood of obtaining the desired mutant, either selecting against wild-type DNA (e.g., T. Kunkel, U.S. Pat. No. 4,873,192 (1989), incorporated herein by reference), or for the mutated DNA (e.g., "Altered Sites (TM) In Vitro Mutagenesis System Technical Manual", Promega Corporation (1990), incorporated herein by reference). A commercial kit for polymerase III site-directed mutagenesis is commercially available from Stratagene, La Jolla, Calif. under the tradename MUTATOR.TM. (Catalogue #200500).
The most general method for mutant screening is by hybridizing DNA from cells transformed with the double-stranded mutagenesis product with a 5'-labeled mutagenic oligonucleotide (R. Wallace et al., Science 209:1396 (1980), incorporated herein by reference). Under nonstringent conditions (e.g., room temperature wash) the probe hybridizes both to the mutant DNA to which it is perfectly matched and also to wild-type DNA to which it is mismatched. By increasing the stringency of washing (e.g., by elevating the temperature) the mutagenic oligonucleotide can be selectively dissociated from wild-type DNA, leaving it bound to mutant DNA. DNA is then prepared and sequenced to verify the mutation. If one employs oligonucleotide-directed mutagenesis methods which result in a high proportion of transformed cells bearing mutant DNA, one can often use sequencing itself as a mutant screen.
Although oligonucleotide-directed mutagenesis has become a valuable tool in the hand of biologists, it remains time-consuming and expensive to perform, particularly when large numbers of mutations or multiple mutations on the same template are required. F

REFERENCES:
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patent: 5256770 (1993-10-01), Glaser et al.
Tsurushita et al., "Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme" Gene (1988) 62: 135-139.
Vandeyar et al., "A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants" Gene (1988) 65:129-133.
Zoller et al., "Oligonucleotide-directed mutagenesis using M-13 derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA" Nucleic Acids Research (1982) 10: 6487-6500.
Wallace et al., "Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA" Nucleic Acids Research (1981) 9:3647-3656.
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D. Young-Sharp et al. "Site-Directed Mutagenesis Using Three Primers and Diagnostic RFLPs In A Single-Dtep Polymerase Chain Reaction," Technique--A Journal of Methods in Cell and Molecular Biology, Jun. 1990, vol. 2, No. 3, pp. 155-162.
J. Liu et al. "An Efficient Method for Introducing Block Mutations Into Specific Regions of a Gene," Biotechniques (1990) 9:738-742.
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T-J. Shen et al. "A marker-coupled method for site-directed mutagenesis," Gene (1991) 103:73-77.

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