Relating to mutagenesis of nucleic acids

Details Relating to mutagenesis of nucleic acids Relating to mutagenesis of nucleic acids

1998-07-06

2000-11-28

Crane, L. Eric

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 2623, 536 2626, 435 912, 435 911, C07H 2102, C07H 2104, C12P 1934

Patent

active

061537454

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to certain novel compounds, a method of mutating a nucleic acid sequence involving the novel compound, and to a kit for performing the method of the invention.


BACKGROUND OF THE INVENTION

In vitro site-directed mutagenesis, which involves the substitution of single amino acids in a protein by changing the relevant base residues in the encoding DNA, has proved to be a powerful method in protein engineering. This technique typically requires information on the structure-function relationship of the protein under study in order to provide a rationale for generating mutants with altered properties. In contrast, random mutagenesis of the DNA region of interest coupled with adequate screening or selection procedures provides an alternative and general method for the generation of DNA, RNA or protein species with improved or novel functions in the absence of initial structural information.
Several methods for the generation of mutants of large DNA fragments have been described and involve using pools of random sequence synthetic oligonucleotides (Matteucci & Heyneker, Nucl. Acids Res. 1983 11, 3113; Wells et al., Gene 1985 34, 315; Nerr et al., DNA 1988 7, 127 and references therein), chemical modification of the target sequence (Kadonaga & Knowles, Nucl. Acids Res. 1985 13, 1733; Meyers et al, Science 1985 229, 242, and references described therein); or base misincorporation using an error-prone polymerase (Lehtovaara et al, Protein Eng. 1988 2, 63).
The synthetic oligonucleotide approach is restricted by the length of the DNA amenable to chemical synthesis, whilst the chemical approach is often labour intensive. In other approaches, random mutations are generated using the polymerase chain reaction (PCR). One such method relies exclusively on the intrinsic error frequency of Taq DNA polymerase, resulting in about 0.5.times.10.sup.-3 mutations per base pair (Zhou, Nucl. Acids Res. 1991 19, 6052). In an improved variation of this method the target sequence of interest is copied under conditions which further reduce the fidelity of DNA synthesis catalysed by Taq DNA polymerase e.g. by the addition of the cofactor manganese and by the use of high concentrations of magnesium and the relevant deoxynucleoside triphosphates (dNTPs--see Leung et al., Techniques 1989 1, 11). Using the latter procedure mutation frequencies in the order of 20.times.10.sup.-3 mutations per base pair have been claimed.
An alternative approach to PCR-based random mutagenesis is to replace, partially or fully, the 5'-triphosphates of the four natural nucleosides by the triphosphates of nucleoside analogues which display ambivalent base pairing potential. To our knowledge this approach has only been attempted using deoxyinosine triphosphate--dITP (Spee et al, Nucl. Acids Res. 1993 21, 777; Ikeda et al, J. Biol. Chem. 1992 267, 6291). However, this analogue is a poor substrate for Taq Polymerase and cannot support DNA synthesis when replacing any of the four normal dNTPs. As a result, four separate PCR reactions are required containing dITP and three dNTPs in equal concentrations together with limiting concentrations of the fourth dNTP. The four separate PCR products are then pooled and cloned (Spee et al., cited above).
A general feature of the above procedures is that the yield of mutant sequences is low and that the pattern of mutations is heavily biased towards transitions (pyrimidine-pyrimidine or purine-purine substitutions). In addition, with the last two methods, undesirable base additions or deletions occur at an appreciable rate.
In an alternative approach, it was envisaged that the 5'-triphosphates of a pyrinidine or purine nucleoside analogue capable of inducing transition mutations in combination with other triphosphate analogues capable of causing transversion mutations would allow efficient random mutagenesis via PCR. The nucleoside analogues P (Kong Thoo Lin & Brown, Nucl. Acids Res. 1989 17, 10373) and K (Brown & Kong Thoo Lin, Carbohydrate Res. 1991 216, 129), (structures 1 and

REFERENCES:
patent: 5434257 (1995-07-01), Matteucci et al.
Brown et al., "Synthesis and Duplex Stability of Oligonucleotides Containing Adenine-Guanine Analogues," Carbohydrate Research, 216, 129-139 (Sep. 2, 1991).
Thoo-Lin et al.(I), "Synthesis and Duplex Stability of Oligonucleotides Containing Cytosine-Thymine Analogues," Nucleic Acids Research, 17(24), 10373-10383 (Dec. 25, 1989).
Nedderman et al.(I), "Base Pairing of Cytosine Analogues with Adenine and Guanine in Oligonucleotide Duplexes: Evidence for Exchange Between Watson-Crick and Wibble Base Pair Using .sup.1 H NME Spectroscopy," J. Chemical Society, Chemical Communications, (Issue No. 18), 1357-1359 (Oct. 1991).
Loakes et al., "The Synthesis of Bicyclic N.sup.4 -Amino-2'-deoxycytidine Derivatives," Nucleosides and Nucleotides,14(3-5), 1057-1059 (May-Jul. 1995).
Van Meervelt et al., "To Wobble or Not to Wobble: Modified Bases Incorporated into DNA," Nucleosides and Nucleotides, 14(3-5), 1057-1059 (May-Jul. 1995).
Nedderman et al.,(i), "Molecular Basis for Methoxyamine-Initiated Mutagenesis: Proton Nuclear Magnetic Resonance Studies of Oligonucleotide Duplexes Containing Base-Modified Cystosine Residues," J. Molecular Biology, 230(3), 1068-1076 (1993); Chem. Abstr., 119(5), p. 320, Abstr. No. 43094b (Aug. 2, 1993); only Abstract supplied.
Thoo-Lin et al.(II), "Synthesis of Oligodeoxyribonucleotides Containing Degenerate Bases and Their Use as Primers in the Polymerase Chain Reaction," Nucleic Acids Research, 20(19), 5149-5152 (Oct. 11, 1992).
Abdul-Masih et al., "Biochemical Studies on the Mutagen, 6-N-Hydroxylaminopurine," J. Biological Chemistry, 261, 2020-2026 (Feb. 15, 1966).
Abdul-Masih, "Biochemical Studies ont he Synthesis of 6-N-Hydroxypurine Deoxynucleoside Triphosphate and Its Incorporation Into Deoxyribonucleic Acid," University Microfilms Document No. 8518469, 1985, Ph.D Dissertation (John Hopkins University, Baltimore MD, University Microfilms International, Ann Arbor, MI.
Budowsky et al.(I), "Mechanism of Mutagenic Action of Hydroxylamine. II. Reaction of Hydroxylamine with the Adenine Nucleus," J. Molecular Biology, 44, 205-207 (Sep. 28, 1969).
Singer et al., "Reaction of O-Methylhydroxylamine with Adenosine Shifts Tautomeric Equilibrium to Cause Transitions," FEBS Letters, 139(1), 69-71 (Mar. 8, 1982).
Budowsky et al.(II), "Mechanism of Mutagenic Action of Hydroxylamine. VIII. Functional Properties of the Modified Adenosine Residues," Biochimica et Biophysica Acta, 390(1), 1-13 (April 16, 1975).
Janion, "The Synthesis and Properties of N.sup.6 -Substituted 2-Amino purine Derivatives," Acta Biochimica Polonica, 23(1), 57-67 (1976).
Nakao et al.(Takeda Chemical Industries), "6-Hydroxylaminopurine Ribotides by Bacterial Fermentation," Japanese Patent Publication 72 08 587, Sep. 21, 1968; Chem. Abstr., 77(5), p. 376, Abstr. No. 32695m (Jul. 31, 1972); only Abstract supplied.
Donaldson et al., "Synthetic and Regulatory Properties of an Adenosine 5'-Phosphate Analog, N.sup.6 -Methoxyadenosine 5'-Phosphate," Biochimica et Biophysica Acta, 184(3), 655-657 (1969.dagger.); Chem. Abstr., 71(19), p. 168, Abstr. No. 89293j (Nov. 10, 1969); only Abstract supplied.
Tindall et al., "Fidelity of DNA Synthesis by the Thermus aquaticus DNA Polymerase," Biochemistry, 27(16), 6008-6013 (Aug. 9, 1988).
Zaccolo et al., "An Approach to Random Mutagenesis of DNA Using Mixtures of Triphosphate Derivatives on Nucleoside Analogues," J. Molecular Biology, 255, 589-603 (1996.dagger.).
Moore et al., "Direct Observation of Two Base Pairing Modes of a Cytosine-Thymine Analogue with Guanine in a DNA Z-form Duplex: Significance for Base Analogue Mutagenesis," Journal of Molecular Biology, 251(1), 665-673 (Sep. 1, 1995).

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