DNA polymersases with enhanced length of primer extension

Details DNA polymersases with enhanced length of primer extension DNA polymersases with enhanced length of primer extension

1997-09-16

2002-06-25

Campbell, Eggerton A. (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120

Reexamination Certificate

active

06410277

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is directed to DNA polymerases, and more particularly, to a novel formulation of DNA polymerases, which formulation of enzymes is capable of efficiently catalyzing the amplification by PCR (the polymerase chain reaction) of unusually long and faithful products.
DNA polymerase obtained from the hot springs bacterium
Thermus aquaticus
(Taq DNA polymerase) has been demonstrated to be quite useful in amplification of DNA, in DNA sequencing, and in related DNA primer extension techniques because it is thermostable. Thermostable is defined herein as having the ability to withstand temperatures up to 95° C. for many minutes without becoming irreversibly denatured, and the ability to polymerize DNA at high temperatures (60° to 75° C.). The DNA and amino acid sequences described by Lawyer et al., J. Biol. Chem. 264:6427 (1989), GenBank Accession No. J04639, define the gene encoding
Thermus aquaticus
DNA polymerase and the enzyme
Thermus aquaticus
DNA polymerase as those terms are used in this application. The highly similar DNA polymerase (Tfl DNA polymerase) expressed by the closely related bacterium
Thermus flavus
is defined by the DNA and amino acid sequences described by Akhmetzjanov, A. A., and Vakhitov, V. A. (1992) Nucleic Acids Research 20:5839, GenBank Accession No. X66105. These enzymes are representative of a family of DNA polymerases, also including
Thermus thermophilus
DNA polymerase, which are thermostable. These enzymes lack a 3′-exonuclease activity such as that which is effective for editing purposes in DNA polymerases such as
E. coli
DNA polymerase I, and phages T7, T3, and T4 DNA polymerases.
Gelfand et al., U.S. Pat. No. 4,889,818 describe a wild-type (abbreviation used here: WT), native
Thermus aquaticus
DNA polymerase. Gelfand et al., U.S. Pat. No. 5,079,352 describe a recombinant DNA sequence which encodes a mutein of
Thermus aquaticus
DNA polymerase from which the N-terminal 289 amino acids of
Thermus aquaticus
DNA polymerase have been deleted (claim 3 of '352, commercial name Stoffel Fragment, abbreviation used here: ST), and a recombinant DNA sequence which encodes a mutein of
Thermus aquaticus
DNA polymerase from which the N-terminal 3 amino acids of
Thermus aquaticus
DNA polymerase have been deleted (claim 4 of '352, trade name AmpliTaq, abbreviation used here: AT). Gelfand et al. report their muteins to be “fully active” in assays for DNA polymerase, but data as to their maximum thermostability is not presented.
Amplification of DNA spans by the polymerase chain reaction (PCR) has become an important and widespread tool of genetic analysis since the introduction of thermostable Taq DNA polymerase for its catalysis. However, one remaining limitation to prior art methods of PCR is the size of the product span that can be amplified. For full-length Taq DNA Polymerase and for N-terminally truncated variants such as Klentaq-278, Klentaq5 and Stoffel Fragment, PCR amplification apparently rapidly becomes inefficient or non-existent as the length of the target span exceeds 5-6 kb. This was shown even when 30 minutes was used during the extension step of each cycle.
Although there are several reports of inefficient but detectable amplification at 9-10 kb target length and one at 15 kb, most general applications are limited to 5 kb.
Kainze et al. (Analytical Biochem. 202:46-49(1992)) report a PCR amplification of over 10 kb: a 10.9 kb and a 15.6 kb product, utilizing an enzyme of unpublished biological source (commercially available as “Hot Tub” DNA polymerase). Kainze et al. report achieving a barely visible band at 15.6 kb after 30 cycles, starting with 1 ng of &lgr; DNA template per 100 ul of reaction volume. The efficiency of this amplification was shown to be relatively low, although a quantitative calculation of the efficiency was not presented. Attempts by Kainze et al. to make WT
Thermus aquaticus
DNA polymerase perform in the 10-15 kb size range were not successful, nor have successful results been reported by anyone else for any form of
Thermus aquaticus
DNA polymerase in this size range.
A DNA polymerase formulation capable of efficient amplification of DNA spans in excess of 6 kb would significantly expand the scope of applications of PCR. For instance, whole plasmids, and constructs the size of whole plasmids, could be prepared with this method, which would be especially valuable in cases in which a portion of the DNA in question is toxic or incompatible with plasmid replication when introduced into
E. coli
. If this thermostable DNA polymerase preparation simultaneously conferred increased fidelity to the PCR amplification, the resulting large products would be much more accurate, active and/or valuable in research and applications, especially in situations involving expression of the amplifed sequence. If the thermostable DNA polymerase preparation allowed, in addition, more highly concentrated yields of pure product, this would enhance the method of PCR to the point where it could be used more effectively to replace plasmid replication as a means to produce desired DNA fragments in quantity.
SUMMARY OF THE INVENTION
Among the several objects of the invention, therefore, may be noted the provision of a formulation of DNA polymerases capable of efficiently catalyzing primer extension products of greater length than permitted by conventional formulations, including lengths up to at least 35 kilobases, that reduces the mutagenicity generated by the PCR process, particularly in comparison with prior art DNA polymerases and for any target lengths, that maximizes the yield of PCR target fragments and, concomitantly, enhances the intensity and sharpness of PCR product bands, without significant sacrifice in flexiblity, specificity, and efficiency; and the provision of an improved process for amplification by PCR which can be utilized to reliably synthesize nucleic acid sequences of greater length and which can effectively utilize PCR products as primers.
Briefly, therefore, the present invention is directed to a novel formulation of thermostable DNA polymerases including at least one thermostable DNA polymerase lacking 3′-5′ exonuclease activity and at least one thermostable DNA polymerase exhibiting 3′-5′ exonuclease activity.
In another aspect, a kit for the synthesis of a polynucleotide is provided, comprising a first DNA polymerase which posesses 3′-5′ exonuclease activity, and a second DNA polymerase which lacks 3′-5′ exonuclease activity.
In another aspect, a kit for the synthesis of a polynucleotide is provided, comprising a first DNA polymerase which posesses 3′-5′ exonuclease activity, and a second DNA polymerase which lacks 3′-5′ exonuclease activity, wherein the first DNA polymerase is selected from the group consisting of
Pyrococcus furiosus
DNA polymerase,
Thermotoga maritima
DNA polymerase,
Thermococcus litoralis
DNA polymerase, and Pyrococus GB-D DNA polymerase, and the second DNA polymerase is selected from the group consisting of
Thermus aquaticus
DNA polymerase, (exo−)
Thermococcus literalis
DNA polymerase, (exo−)
Pyrococcus furiosus
DNA polymerase, and (exo−) Pyrococcus GB-D DNA polymerase.
In a further embodiment of the invention, a method of amplifying a polynucleotide sequence is provided. The method includes the steps of mixing a composition with a synthesis primer, and a synthesis template, with the composition including a first DNA polymerase possessing 3′-5′ exonuclease activity, and a second DNA polymerase lacking 3′-5′ exonuclease activity.
In yet another aspect of the invention, a method of amplifying a polynucleotide sequence is provided. The method includes the steps of mixing a composition with a synthesis primer, and a synthesis template, with the composition including a first DNA polymerase possessing3′-5′ exonuclease activity which is selected from the group consisting of
Pyrococcus furiosus
DNA

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