Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-06-17
2002-11-12
Prouty, Rebecca E. (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S193000, C435S006120, C435S091200, C530S350000, C530S358000
Reexamination Certificate
active
06479267
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The instant disclosure pertains to thermostable DNA polymerases which exhibit improved robustness and efficiency.
2. Background
DNA polymerases are enzymes which are useful in many recombinant DNA techniques such as nucleic acid amplification by the polymerase chain reaction (“PCR”), self-sustained sequence replication (“3SR”), and high temperature DNA sequencing. Thermostable polymerases are particularly useful. Because heat does not destroy the polymerase activity, there is no need to add additional polymerase after every denaturation step.
However, many thermostable polymerases have been found to display a 5′ to 3′ exonuclease or structure-dependent single-stranded endonuclease (“SDSSE”) activity which may limit the amount of product produced or contribute to the plateau phenomenon in the normally exponential accumulation of product. Such 5′ to 3′ nuclease activity may contribute to an impaired ability to efficiently generate long PCR products greater than or equal to 10 kb, particularly for G+C rich targets. In DNA sequencing applications and cycle sequencing applications, the presence of 5′ to 3′ nuclease activity may contribute to a reduction in desired band intensities and/or generation of spurious or background bands.
Additionally, many of the enzymes presently available are sensitive to high salt environments and have low processing ability, that is, the number of nucleotides incorporated per DNA polymerase binding event. Furthermore, addition of dITP to the reaction mixture to address compression problems usually results in reduced activity of the enzyme.
Thus, a need continues to exist for an improved DNA polymerase having increased tolerance to high salt conditions, efficient utilization of dITP, high productivity, and improved performance on GC-rich templates.
BRIEF SUMMARY OF THE INVENTION
The instant disclosure teaches a purified recombinant thermostable DNA polymerase comprising the amino acid sequence set forth in
FIG. 1
, as well as a purified recombinant thermostable DNA polymerase which exhibits at least about 80% activity at salt concentrations of 50 mM and greater. The instant disclosure further teaches a purified recombinant thermostable DNA polymerase which exhibits at least about 70% activity at salt concentrations of 25 mM and greater, and a purified recombinant thermostable DNA polymerase having a processivity of about 30 nucleotides per binding event.
The instant disclosure also teaches an isolated nucleic acid that encodes a thermostable DNA polymerase, wherein said nucleic acid consists of the nucleotide sequence set forth in
FIG. 1
, as well as a recombinant DNA vector that comprises the nucleic acid, and a recombinant host cell transformed with the vector.
The instant disclosure also teaches a method of sequencing DNA comprising the step of generating chain terminated fragments from the DNA template to be sequenced with the DNA polymerase in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and determining the sequence of said DNA from the sizes of said fragments. The instant disclosure also teaches a kit for sequencing DNA comprising the DNA polymerase.
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Takagi et al. Characterization of DNA Polymerase from Pyrococcus sp. Strain KOD1 and its Application to PCR, Applied and Enviromental Microbiology 63(11): 4504-4510, Nov. 1997.*
Gutman, Pablo and Minton, Kenneth; Conserved Sites in the 5′-3′ Exonuclease Domain ofEscherichia coliDNA Polymerase; Nucleic Acids Research, 1993, vol. 21, No. 18, pp. 4406-4407.
Reeve, Michael and Fuller, Carl; A Novel Thermostable Polymerase for DNA Sequencing; Nature, vol. 376, Aug. 31, 1995, pp. 796-797.
Davis Maria Cuozzo
Fuller Carl W.
Huang Lin
Mamone Joseph Anthony
Amersham Pharmacia Biotech Inc.
Hutson Richard
Prouty Rebecca E.
Ronning, Jr. Royal N.
Ryan Stephen G.
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