Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1997-11-26
2002-05-28
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S091200
Reexamination Certificate
active
06395524
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to thermostable polymerases and more specifically to methods for identifying polymerase mutants having desired fidelity.
Every living organism requires genetic material, deoxyribonucleic acid (DNA), to pass a unique collection of characteristics to its offspring. Genes are discreet segments of the DNA and provide the information required to generate a new organism. Even simple organisms, such as bacteria, contain thousands of genes, and the number is many fold greater in complex organisms such as humans. Understanding the complexities of the development and functioning of living organisms requires knowledge of these genes. However, the amount of DNA that can be isolated for study has often been limiting.
A major breakthrough in the study of genes was the development of the polymerase chain reaction (PCR). PCR amplifies genes or portions of genes by making many identical copies, allowing isolation of genes from very tiny amounts of DNA. The motors for PCR are DNA polymerases that copy the DNA of each gene during each round of DNA synthesis. Using oligonucleotides that determine the start and termination of DNA synthesis, a single gene can be replicated into millions of copies. This process has created a revolution in biotechnology and has been used extensively for the identification of mutant genes that are responsible for or associated with inherited human diseases. It is now possible to identify a mutant gene in a single cell, amplify the gene a million times, and establish the nature of the mutation. One application of identifying a mutant gene is the determination of genetic susceptibility to disease, which can be mapped by gene amplification and DNA sequencing.
DNA polymerases function in cells as the enzymes responsible for the synthesis of DNA. They polymerize deoxyribonucleoside triphosphates in the presence of a metal activator, such as Mg
2+
, in an order dictated by the DNA template or polynucleotide template that is copied. Even though the template dictates the order of nucleotide subunits that are linked together in the newly synthesized DNA, these enzymes also function to maintain the accuracy of this process. The contribution of DNA polymerases to the fidelity of DNA synthesis is mediated by two mechanisms. First, the geometry of the substrate binding site in DNA polymerases contributes to the selection of the complementary deoxynucleoside triphosphates. Mutations within the substrate binding site on the polymerase can alter the fidelity of DNA synthesis. Second, many DNA polymerases contain a proof-reading 3′-5′ exonuclease that preferentially and immediately excises non-complementary deoxynucleoside triphosphates if they are added during the course of synthesis. As a result, these enzymes copy DNA in vitro with a fidelity varying from 5×10
−4
(1 error per 2000 bases) to 10
−7
(1 error per 10
7
bases) (Fry and Loeb,
Animal Cell DNA Polymerases,
pp. 221, CRC Press, Inc., Boca Raton, Fla. (1986); Kunkel, T. A.,
J. Biol. Chem.
267:18251-18254(1992)).
In vivo, DNA polymerases participate in a spectrum of DNA synthetic processes including DNA replication, DNA repair, recombination, and gene amplification (Kornberg and Baker,
DNA Replication,
pp. 929, W. H. Freeman and Co., New York (1992)). During each DNA synthetic process, the DNA template is copied once or at most a few times to produce identical replicas. In vitro DNA replication, in contrast, can be repeated many times, for example, during PCR.
In the initial studies with PCR, the DNA polymerase was added at the start of each round of DNA replication. Subsequently, it was determined that thermostable DNA polymerases could be obtained from bacteria that grow at elevated temperatures, and these enzymes need to be added only once. At the elevated temperatures used during PCR, these enzymes would not denature. As a result, one can carry out repetitive cycles of polymerase chain reactions without adding fresh enzymes at the start of each synthetic addition process. The commercial market for the sale of DNA polymerases from thermostable organisms can be conservatively estimated at 200 million dollars per year. DNA polymerases, particularly thermostable polymerases, are the key to a large number of techniques in recombinant DNA studies and in medical diagnosis of disease.
Due to the importance of DNA polymerases in biotechnology and medicine, it would be highly advantageous to generate DNA polymerases having desired enzymatic properties such as altered fidelity. However, the ability to predict the effect of introducing an amino acid mutation into the sequence of a protein remains very limited. Even when structural information is available for the protein of interest, it is often very difficult to predict the effect of mutations of specific amino acid residues on the function of that protein. In particular, it is extremely difficult to predict amino acid substitutions that will alter the activity of an enzyme to achieve a desirable change.
Despite the limitations in predicting the effect of introducing amino acid substitutions into proteins, a number of mutant DNA polymerases have been discovered, or have been created by site-specific mutagenesis, and have been used in PCR amplification (Tabor and Richardson,
Proc. Natl. Acad. Sci. USA
20 92:6339-6343 (1995)). Some of these mutant polymerases offer particular advantages with respect to thermostability, processivity, length of the newly synthesized DNA product, or fidelity of DNA synthesis. Those that are more accurate for the most part contain a 3′-5′ exonuclease activity that removes misincorporated bases prior to adding the next nucleotide during DNA synthesis. However, the current spectrum of mutant DNA polymerases is quite limited. For the most part, these mutants have been obtained by introducing a single base substitution at a specified site, purifying the enzyme and studying the changes in catalytic activity (Joyce and Steitz,
Annu. Rev. Biochem.
63:777-822 (1994)). These laborious and step-wise procedures have been necessary due to the lack of adequate knowledge to predict the effects of most single amino acid substitutions and due to the lack of rules for predicting the effects of multiple simultaneous substitutions.
Thus, there exists a need for rapid and efficient methods to produce and screen for modified polymerases having desired fidelity in polynucleotide synthesis. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides a method for identifying a thermostable polymerase having altered fidelity. The method consists of generating a random population of polymerase mutants by mutating at least one amino acid residue of a thermostable polymerase and screening the population for one or more active polymerase mutants by genetic selection. For example, the invention provides a method for identifying a thermostable polymerase having altered fidelity by mutating at least one amino acid residue in an active site O-helix of a thermostable polymerase. The invention also provides thermostable polymerases and nucleic acids encoding thermostable polymerases having altered fidelity, for example, high fidelity polymerases and low fidelity polymerases. The invention additionally provides a method for identifying one or more mutations in a gene by amplifying the gene with a high fidelity polymerase. The invention further provides a method for accurately copying repetitive nucleotide sequences using a high fidelity polymerase mutant. The invention also provides a method for diagnosing a genetic disease using a high fidelity polymerase mutant. The invention further provides a method for randomly mutagenizing a gene by amplifying the gene using a low fidelity polymerase mutant.
REFERENCES:
patent: 5614365 (1997-03-01), Tabor et al.
patent: 5939292 (1999-08-01), Gelfand et al.
patent: 5945312 (1999-08-01), Goodman et al.
patent: 5948614 (1999-09-01), Chatterjee
patent
Hood Leroy
Loeb Lawrence A.
Suzuki Motoshi
Campbell & Flores LLP
Houtteman Scott W.
University of Washington
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