Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-03-12
2001-07-24
Prouty, Rebecca E. (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S006120, C536S063000
Reexamination Certificate
active
06265193
ABSTRACT:
FIELD OF THE INVENTION
The invention is related to DNA polymerases having mutations that alter the ability of the enzyme to incorporate labeled nucleotides into a polynucleotide molecule.
BACKGROUND
DNA polymerases are enzymes that synthesize the formation of DNA molecules from deoxynucleotide triphosphates using a template DNA strand and a complementary synthesis primer annealed to a portion of the template. A detailed description of DNA polymerases and their enzymological characterization can be found in Kornberg,
DNA Replication Second Edition,
W. H. Freeman (1989).
DNA polymerases have a variety of uses in molecular biology techniques suitable for both research and clinical applications. Foremost among these techniques are DNA sequencing and nucleic acid amplification techniques such as PCR (polymerase chain reaction).
The amino acid sequence of many DNA polymerases have been determined. Sequence comparisons between different DNA polymerase have identified many regions of homology between the different enzymes. X-ray diffraction studies have determined the tertiary structures of Klenow fragment, T7 DNA polymerase, and Taq DNA polymerase. Studies of the tertiary structures of DNA polymerases and amino acid sequence comparisons have revealed numerous structural similarities between diverse DNA polymerases. In general, DNA polymerases have a large cleft that is thought to accommodate the binding of duplex DNA. This cleft is formed by two sets of helices, the first set is referred to as the “fingers” region and the second set of helices is referred to as the “thumb” region. The bottom of the cleft is formed by anti-parallel &bgr; sheets and is referred to as the “palm” region. Reviews of DNA polymerase structure can be found in Joyce and Steitz,
Ann. Rev. Biochem.
63:777-822 (1994). Computer readable data files describing the three-dimensional structure of some DNA polymerases have been publicly disseminated.
Fluorescently labeled nucleotides have greatly simplified and improved the utility of many procedures in molecular biology. The use of fluorescently labeled nucleotides for labeling polynucleotides in synthesis procedures, has to a large extent replaced the use of radioactive labeling. Fluorescently labeled nucleotides have been widely used in DNA sequencing, see Smith et al Nature 321:674-679 (1986), in PCR, and other forms of polynucleotide fragment analysis.
A major problem with using fluorescently labeled nucleotides is the ability of DNA polymerases to discriminate against the incorporation of fluorescently labeled nucleotides. For example, the inventors have discovered that in competition assays between a TET (6-carboxy-4,7,2′,7′-tetrachlorofluorescein) labeled 2′3′ dideoxynucleotide and the corresponding unlabeled dideoxynucleotide, Taq DNA polymerase incorporates the unlabeled dideoxynucleotide into DNA at least 85 times more frequently than the corresponding unlabeled nucleotide. This discrimination between labeled and unlabeled nucleotides has profound effects on procedures using DNA polymerases to label DNA. For example, much larger amounts of fluorescently labeled nucleotide must be used in sequencing reactions. This large amount of fluorescently labeled nucleotide is expensive and can generate excessive background fluorescence, thereby reducing the yield of sequence information.
In view of the problems arising from the ability of DNA polymerases to discriminate against the incorporation of fluorescently labeled nucleotides, the inventors have developed several novel DNA polymerases that have reduced discrimination against the incorporation of one or more fluorescently labeled nucleotides into DNA.
SUMMARY
Naturally occurring DNA polymerases preferentially incorporate unlabeled nucleotides over corresponding fluorescently labeled nucleotides into polynucleotides. This ability of DNA polymerases to discriminate against fluorescently labeled nucleotide has undesirable effects on many molecular biology procedures that require the enzymatic addition of fluorescently labeled nucleotides, e.g., labeled dideoxy terminator sequencing. The present invention relates to mutant DNA polymerases that exhibit reduced discrimination against fluorescently labeled nucleotides into polynucleotides.
The DNA polymerases of the invention have at least one mutation in the nucleotide label interaction region of the enzyme such that the mutation results in reduced discrimination against fluorescently labeled nucleotides. The nucleotide label interaction region of a DNA polymerase is formed by portions of the O-helix, (ii) the K helix, and (iii) the inter O-P helical loop of Taq DNA polymerase or analogous positions in other DNA polymerases. Amino acid residues within the nucleotide label interaction region as defined by TET (II).ddC are E520. A531, L522, R523, E524, A525, H526, P527,I528, V529, E530, K531, I532, R536, E537, R573, Q582, N583, V586, R587, P589, Q592, R593, R595, D610, T612, Q613, E615, R636, D637, T640, F647, V654, D655, P656, L657, R659, R660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832. The sites at R660, T664, and E681 are of prefered sites for introducing mutations. In a preferred embodiment of the invention for use with fluorescein-type dyes, a mutation is present at position 681 converting an E (glutamic acid) to M (methionine), i.e., E681M. In a preferred embodiment of the invention for use with fluorescein- fluorescein energy transfer dyes a mutation is present at position 657 converting an L (leucine) to a G (glycine). In addition to providing mutant Taq DNA polymerases having reduced discrimination against labeled nucleotides, the invention includes mutants derived from a wide variety of DNA polymerases, both thermostable and otherwise.
In addition to providing novel mutant DNA polymerases, the invention also provides polynucleotides encoding the subject mutant DNA polymerases. The polynucleotides provided may comprise expression vectors for the recombinant production of the mutant polymerases. The invention also includes host cells containing the subject polymerase polynucleotides.
The invention also includes numerous methods of using the subject DNA polymerases. The subject methods involve synthesizing a fluorescently labeled polynucleotide by means of a polynucleotide synthesis reaction catalyzed by a mutant DNA polymerase that has reduced discrimination against incorporating labeled nucleotides into polynucleotides. The subject methods of polynucleotide synthesis include the step of extending a primed polynucleotide template with at least one fluorescent labeled nucleotide, wherein the extension is catalyzed by a DNA polymerase that has reduced discrimination against labeled nucleotides into polynucleotides. The subject methods of synthesizing a fluorescently labeled polynucleotide may be used in a variety of methods such as Sanger sequencing and the polymerase chain reaction (PCR).
Another aspect of the invention is to provide kits for synthesizing fluorescently labeled polynucleotides in accordance with the methods of the invention. Kits of the invention comprise a mutant DNA polymerase of the invention and a fluorescently labeled nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerase in the kit.
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Kalman et al., “Thermostable DNA Polymerases with Altered Discrimination Properties,”Genome Sci. Technol• 1:42 (Abstract Only, A-14) (1995).
Reeve et al., “A novel thermostable polymerase for DNA Sequencing,”Nature 376:796-797(Aug. 31, 1995).
Suzuki et al., “Random mutagenesis ofThermus aquaticusDNA polymerase I:Concordance of immutable sites in vivo with the c
Bloom Curtis
Brandis John
Richards John H.
Bortner Scott R.
Hutson Richard
PE Corporation (NY)
Prouty Rebecca E.
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