Mutant chimeric DNA polymerase

Details Mutant chimeric DNA polymerase Mutant chimeric DNA polymerase

1998-06-26

2001-05-08

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S252300, C435S254110, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06228628

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a mutant chimeric thermostable DNA polymerase, methods for its synthesis, and methods for its use. The enzyme is useful in many recombinant DNA techniques, especially nucleic acid sequencing and nucleic acid amplification by the polymerase chain reaction (PCR).
2. Background Art
Thermostable DNA polymerases, which catalyze the template-directed polymerization of deoxyribonucleoside triphosphates (dNTPs) to form DNA, are used in a variety of in vitro DNA synthesis applications, such as DNA sequencing and DNA amplification. Typically, naturally occurring DNA polymerases strongly discriminate against the incorporation of nucleotide analogues. This property contributes to the fidelity of DNA replication and repair. However, the incorporation of nucleotide analogues is useful for many DNA synthesis applications, in particular, in DNA sequencing.
DNA sequencing reactions using the chain termination method initially described by Sanger et al., 1977
, Proc. Natl. Acad. Sci
. 74:5463-5467, incorporated herein by reference, rely on an unconventional substrate, dideoxynucleoside triphosphate (ddNTP), for termination of synthesis. In the chain termination method, both the DNA polymerase's conventional substrate (dNTP) and a chain-terminating, unconventional substrate (ddNTP or labeled ddNTP) are present in the reaction. Synthesis proceeds until a ddNTP is incorporated. To insure that the chain-terminating ddNTPs are incorporated at a suitable rate, the inherent discrimination of the previously utilized DNA polymerases against the incorporation of ddNTPs was overcome by providing an excess of ddNTP.
Dye-terminator sequencing, a variant of the chain termination method, uses ddNTPs labeled with fluorescent dyes, such as fluorescein or rhodamine, to terminate synthesis and, simultaneously, to label the synthesized DNA. The presence of a dye label on the ddNTP can exacerbate the discrimination by the DNA polymerase against the incorporation of the unconventional substrate.
Typically, sequencing by the chain termination method is carried out using repeated steps of primer extension followed by heat denaturation of the primer extension product-template duplex. This embodiment, referred to as cycle sequencing, is carried out in a thermal cycler using a thermostable DNA polymerase. Kits for carrying out cycle sequencing are commercially available from, for example, Perkin Elmer, Norwalk, Conn.
Thermostable DNA polymerases derived from a variety of organisms have been described extensively in the literature and are well known to one of skill in the art. Particular examples include DNA polymerases from a variety of species of the genus Thermus (see U.S. Pat. No. 5,466,591), in particular from
Thermus aquaticus
(Taq DNA polymerase) described in U.S. Pat. Nos. 4,889,818; 5,352,600; and 5,079,352; and the DNA polymerase from
Thermatoga maritima
(Tma DNA polymerase) described in U.S. Pat. Nos. 5,374,553 and 5,420,029; all of which are incorporated herein by reference.
DNA polymerases typically possess one or more associated exonucleolytic activities. For example Tma DNA polymerase catalyzes the exonucleolytic removal of nucleotides from the 5′-end of a double-stranded DNA (referred to as 5′ to 3′ exonuclease activity or 5′-nuclease activity) as well as from the 3′-end of a single- or double-stranded DNA (referred to as 3′ to 5′ exonuclease activity). In contrast, DNA polymerases from the genus Thermus possess only 5′-nuclease activity. A review of thermostable DNA polymerases and their associated activities is found in Abramson, 1995, in PCR Strategies, (Innis et al. ed., Academic Press, Inc.). For use in DNA sequencing, a DNA polymerase that lacks associated exonucleolytic activity, either 5′-nuclease activity or 3′ to 5′ exonuclease activity, is preferred. Mutant forms of a number of thermostable DNA polymerases which lack 5′-nuclease activity are described in U.S. Pat. No. 5,466,591, incorporated herein by reference.
European Patent Application 0 655 506, incorporated herein by reference, describes a mutated DNA polymerase with an enhanced ability to incorporate dideoxynucleotides (see also U.S. Pat. No. 5,614,365, incorporated herein by reference). The mutation is a point mutation corresponding to amino acid 526 of T7 DNA polymerase. Examples of such mutations include mutations in amino acid 667 of Taq DNA polymerase.
AmpliTaq® DNA polymerase, FS, a mutant form of Taq DNA polymerase that has essentially no 5′-nuclease activity and incorporates an F667Y mutation, is sold as a component of DNA cycle sequencing kits by Perkin Elmer, Norwalk, Conn. The F667Y mutation results in a significant reduction in the discrimination against ddNTPs. This property greatly improves the sequencing data obtained from a dye-terminator sequencing reaction and reduces the amount of ddNTPs required for each sequencing reaction. However, the use of AmpliTaq® DNA polymerase, FS has not eliminated problems with non-uniformity of peak heights in the sequencing trace when used with the standard rhodamine dye family-labeled ddNTPs. An analysis of the peak height patterns obtained using AmpliTaq® DNA polymerase, FS in dye-terminator cycle sequencing reactions is described in Parker et al., 1996, BioTechniques 21(4):694-699, incorporated herein by reference.
Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill of the art, are explained fully in the literature. See, for example, Sambrook et al., 1989
, Molecular Cloning—A Laboratory Manual
, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.;
Oligonucleotide Synthesis
(M. J. Gait, ed., 1984);
Nucleic Acid Hybridization
(B. D. Hames and S. J. Higgins. eds., 1984); and a series,
Methods in Enzymology
(Academic Press, Inc.), all of which are incorporated herein by reference. All patents, patent applications, and publications cited herein, both supra and infra, are incorporated herein by reference.
SUMMARY OF THE INVENTION
The present invention relates to mutant, chimeric thermostable DNA polymerases that possess significantly improved properties relative to previously described thermostable DNA polymerases. The DNA polymerase yields substantial improvements when used in DNA sequencing reactions. In particular, the DNA polymerase of the invention provides the following combination of advantageous properties:
1. improved incorporation of ddNTPs;
2. improved uniformity of peak heights in DNA sequencing traces, in particular when used with dye-labeled ddNTPs in a cycle sequencing reaction;
3. reduced rate of pyrophosphorolysis of dye-labeled ddNTPs; and
4. improved incorporation of dITP.
Furthermore, the DNA polymerase can be easily and efficiently expressed to a high level in a recombinant expression system, thereby facilitating commercial production of the enzyme. The combination of properties possessed by the DNA polymerase of the present invention represent a significant advantage over thermostable DNA polymerases previously described in the literature.
The chimeric DNA polymerases of the present invention consist of an N-terminal region derived from the 5′-nuclease domain of a Thermus species DNA polymerase and a C-terminal region derived from the 3′ to 5′ exonuclease and polymerase domains of Tma DNA polymerase. The N-terminal region contains at least a region of the Thermus species DNA polymerase corresponding to amino acids 1-138 of Tma DNA polymerase and may contain up to the entire 5′-nuclease domain of the Thermus species DNA polymerase. The C-terminal region contains, in addition to the 3′ to 5′ exonuclease and polymerase domains of Tma DNA polymerase, a portion of the 5′-nuclease domain of Tma DNA polymerase corresponding to the portion of the 5′-nuclease domain of the Thermus species DNA polymerase not present in the N-terminal region.
Thus, the chimeric DNA polymerase of the present invention cons

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