Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1993-01-05
1995-05-23
Parr, Margaret
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, C12Q 168, C12P 1934
Patent
active
054181496
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to improved methods for amplifying nucleic acids using methods such as the polymerase chain reaction (PCR) procedure. More specifically this invention provides methods to increase the specificity of nucleic acid amplification assays and to minimize the effects of aerosol contamination of nucleic acid amplification reaction assays due to amplified product from previous amplifications. The methods involve the introduction of unconventional nucleotide bases into the amplified product and exposing carryover product to enzymatic (e.g., glycosylases) and/or physical-chemical treatment that effectively render .the product incapable of acting as a template for subsequent amplifications,. This invention also relates to methods for producing nucleic acid-free proteins that are particularly useful as reagents for amplification systems. This invention further relates to efficient means to express uracil-DNA glycosylase and purified preparations of uracil-DNA glycosylase.
DESCRIPTION OF THE PRIOR ART
The incorporation of deoxyuridine into the genome of E. coil has been reported to be especially high in strains having a defective deoxyuridine-triphosphatase. See Sedwick et al., 1986, Mutat. Res. 162(1):7-20. DNA with abasic sites has been prepared by deamidation of cytosine followed by treatment with uracil-DNA glycoslyase. DNA polymerase extension on these templates is terminated by abasic sites. See Sagher and Strauss, 1983, Nucleic Aids Res. 13(12):4285-4298 and Sagher and Strauss, 1983, Biochemistry 22(19):4518-4526. DNA repair enzymes have been reviewed by Sancar and Sancar, 1988, Ann. Rev. Biochem. 57:29-67, and Lindahl, 1982, Ann. Rev. Biochem. 51:61-87.
A method for introducing site-specific mutations into DNA has been described that relies upon replacement of thymine with uracil in DNA and subsequent treatment with uracil-DNA glycosylase. See U.S. Pat. No. 4,873,192 and Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488-492. Uracil-containing phage were suggested as a part of a biological containment system that would transfer generic information only to uracil-N glycosylase deficient cells and not to naturally occurring bacteria. See Warner et al., 1979, J. Biol. Chem. 245 (16):7534-7539.
Contamination from Carryover products is a recognized problem. See Kwok, PCR Protocols (Innis et al. Academic Press 1990), Chapter 17, pages 142-145.
SUMMARY OF THE INVENTION
The present invention provides methods to increase the specificity of nucleic acid amplification processes. The method involves the incorporation of modified nucleotides and a nucleic acid glycosylase specific for the modified nucleotides into the amplification reaction mixture so that non-specific amplification products generated prior to deactivation of the glycosylase are degraded by the glycosylase and rendered incapable of serving as templates for amplification. In amplification processes involving a heat denaturation step to denature double-stranded nucleic acids into single-stranded nucleic acids, the increased specificity provided by the present methods results from degradation of non-specific amplification products generated prior to the first denaturation step.
This invention also provides for methods of "sterilizing" or "restricting" nucleic acid amplification reaction systems contaminated with nucleic acids generated from previous amplifications. The method of the invention involves the generation of nucleic acids that can be rendered incapable of further amplification, so that if amplified nucleic acids produced by the method contaminate an amplification mixture, that contaminated mixture can be treated so that the contaminating nucleic acids are not amplified.
These sterilizing methods comprise: (a) mixing conventional and unconventional nucleotides into an amplification reaction system containing an amplification reaction mixture and a target nucleic acid sequence; (b) amplifying the target nucleic acid sequence to produce amplified products of nucleic acid having the unconventional nucleot
REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
patent: 4683202 (1987-07-01), Mullis
patent: 4873192 (1989-10-01), Kunkel
patent: 4965188 (1990-10-01), Mullis
patent: 5035996 (1991-07-01), Hartley
Chu et al., 1986, Nuc. Acids Res. 14(14):5591-5603, "Synthesis of an Amplifiable Reporter RNA for Bioassays".
Lizardi et al., 1988, Biotechnology 6:1197-1202, "Exponential Amplification of Recombinant-RNA Hybridization Probes".
Wu and Wallace, 1989, Genomics 4:560-569, "The Ligation Amplification Reaction (LAR)-Amplification of Specific DNA Sequences Using Sequential Rounds of Template-Dependent Ligation".
Landegren et al., 1988, Science 241:1077-1080, "A Ligase-Mediated Gene Detection Technique".
Sancar and Sancar, 1988, Ann. Rev. Biiochem. 57:29-67, "DNA Repair Enzymes".
Lindahl, 1982, Ann. Rev. Biochem. 51:61-87, "DNA Repair Enzymes".
Kane and Linn, 1981, J. Biological Chemistry 256(7):3405-3414, "Purification and Characterization of an Apurinic/Apyrimidinic Endonuclease from HeLa Cells".
Verly et al., 1981, Eur. J. Biochem. 118:195-201, "Localization of the Phosphoester Bond Hydrolyzed . . . the Major Apurinic/Apyrimidinic Endodeoxyribonuclease from Rat-Liver Chromatin".
Shaper et al., 1982, J. Biological Chemistry 257(22):13455-13458, "Human Placental Apurinic/Apyrimidinic Endonuclease".
Nakabeppu et al., 1984, J. Biological Chemistry 259(22):13723-13729, "Cloning and Characterization of the alkA Gene of Escherichia coli that Encodes 3-Methyladenine DNA Glycosylase II".
Steinum and Seeberg, 1986, Nuc. Acids Res. 14(9):3763-3773, "Nucleotide Sequence of the tag Gene from Escherichia coli".
Radman, 1976, J. Biological Chemistry 251(5):1438-1445, "An Endonuclease from Escherichia coli That Introduces Single Polynucleotide Chain Scissions in Ultraviolet-Irradiated DNA".
Gates III and Linn, 1977, J. Biological Chemistry 252(5):1647-1653, "Endonuclease V of Escherichia coli".
Gates III and Linn, 1977, J. Biological Chemistry 252(9):2802-2807, "Endonuclease from Escherichia coli That Acts Specifically Upon Duplex DNA Damaged by Ultraviolet LIght, Osmium Tetroxide, Acid, or X-rays".
Demple et al., 1980, Methods of Enzymology 65:224-231, "Purification of Properties of Escherichia coli Endodeoxyibonuclease V".
Varshney et al., 1988, J. Biological Chemistry 263(16:7776-7784, "Sequence Analysis, Expression, and Conservation of Escherichia coli Uracil DNA Glycosylase and Its Gene (ung)".
Morgan and Chlebek, 1989, J. Biological Chemistry 264(17):9911-9914, "Uracil-DNA Glycosylase in Insects".
Duncan et al., 1978, J. Bacteriology 134(3):1039-1045, "Escherichia coli K-12 Mutants Deficient in Uracil-DNA Glycosylase".
Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488-492, "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection".
Bio-Rad Muta-Gene in vitro Mutagenesis Kit Instruction manual, Jul., 1987.
Warner and Duncan, 1978, Nature 272:32-34, "In Vivo Synthesis and Properties of Uracil-Containing DNA".
Warner et al., 1979, J. Biological Chemistry 254(16):7534-7539, "The Properties of a Bacteriophage T5 Mutant Unable to Induce Deoxyuridine 5'-Triphosphate Nucleotidohydrolase".
Sagher and Strauss, 1983, Biochemistry 22:4518-4526, "Insertion of Nucleotides Opposite Apurinic/Apyrimidinic Sites in Deoxyribonucleic Acid During In Vitro Synthesis: Uniqueness of Adenine Nucleotides".
Sagher and Strauss, 1985, Nuc. Acids. Res. 13(12):4285-4298, "Abasic Sites From Cytosine as Termination Signals for DNA Synthesis".
Sedwick et al., 1986, Mutation Research 162(1):7-20, "Deoxyuridine Miscorporation Causes Site-Specific Mutational Lesions in the lacI Gene of Escherichia coli".
Kwok, 1990, PCR Protocols (ed. Innis et al., Academic Press), Chaper 17, pp. 142-145, "Procedures to Minimize PCR-Product Carry-Over".
Sarkar and Sommer, 1990, nature 343:27, "Shedding Light on PCR Contamination".
Kitchin et al., 1990, Nature 344:201, "Avoidance of False Positives".
Diagnostics Report of Sep., 1988.
Longo et al., 1990, Gene 93:125-128, "Use of Uracil DNA Glycosylase to Con
Gelfand David H.
Kwok Shirley Y.
Sninsky John J.
Gould George M.
Hoffmann-La Roche Inc.
Parr Margaret
Petry Douglas A.
Schreiber David
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