Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1993-02-23
1995-11-14
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
935 10, 935 14, 536 232, C12N 912, C12N 1554
Patent
active
054665914
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to thermostable DNA polymerases which have been altered or mutated such that a different level of 5' to 3' exonuclease activity is exhibited from that which is exhibited by the native enzyme. The present invention also relates to means for isolating and producing such altered polymerases. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR) self-sustained sequence replication (3SR), and high temperature DNA sequencing.
2. Background Art
Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al., 1957, J. Biol. Chem. 223:171-177 and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427.
Somewhat less investigation has been made on the isolation and purification of DNA polymerases from thermophiles such as Thermus aquaticus, Thermus thermophilus, Thermotoga maritima, Thermus species sps 17, Thermus species Z05 and Thermosipho africanus. The use of thermostable enzymes to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present was described in U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the PCR process, both disclosures of which are incorporated herein by reference. Primers, template, nucleoside triphosphates, the appropriate buffer and reaction conditions, and polymerase are used in the PCR process, which involves denaturation of target DNA, hybridization of primers, and synthesis of complementary strands. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. The two patents disclose that, if the polymerase employed is a thermostable enzyme, then polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity.
U.S. Pat. No. 4,889,818, European Patent Publication No. 258,017 and PCT Publication No. 89/06691, the disclosures of which are incorporated herein by reference, all describe the isolation and recombinant expression of an .about.94 kDa thermostable DNA polymerase from Thermus aquaticus and the use of that polymerase in PCR. Although T. aquaticus DNA polymerase is especially preferred for use in PCR and other recombinant DNA techniques, there remains a need for other thermostable polymerases.
SUMMARY OF THE INVENTION
In addressing the need for other thermostable polymerases, the present inventors found that some thermostable DNA polymerases such as that isolated from Thermus aquaticus (Taq) display a 5' to 3' exonuclease or structure-dependent single-stranded endonuclease (SDSSE) activity. As is explained in greater detail below, such 5' to 3' exonuclease activity is undesirable in an enzyme to be used in PCR, because it may limit the amount of product produced and contribute to the plateau phenomenon in the normally exponential accumulation of product. Furthermore, the presence of 5' to 3' nuclease activity in a thermostable DNA polymerase 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 applitions, the presence of 5' to 3' nuclease activity may contribute to reduction in desired band intensities and/or generation of spurious or background bands. Finally, the absence of 5' to 3' nuclease activity may facilitate higher sensitivity allelic discrimination in a combined polymerase ligase chain reaction (PLCR) assay.
However, an enhanced or greater amount of 5' to 3' exonuclease activity in a thermostable DNA polymerase may be desirable in such an enzyme which is used in a homogeneous assay system for the concurrent amplification and detection of a target nucleic acid sequence. Generally, an enhanced 5' to 3' exonuclease activity is defined an enhanced rate of exonuclease cleavage or an enhanced rate of nick-tran
REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
patent: 4683202 (1987-07-01), Mullis
patent: 4889818 (1989-12-01), Gelfand et al.
patent: 5079352 (1992-01-01), Gelfand et al.
patent: 5108892 (1992-04-01), Burke et al.
patent: 5210015 (1993-05-01), Gelfand et al.
Bernad et al., 1989, "A Conserved 3'-5' Exonuclease Active Site in Prokaryotic and Eukaryotic DNA Polymerases" Cell 59:219-228.
Lawyer et al., 1989, "Isolation, Characterization, and Expression in Escherichia coli of the DNA Polymerase Gene From Thermus Aquaticus" J. Biol. Chem 264(11):6427-6437.
Leavitt and Ito, 198, "T5 DNA Polymerase: Structural-Functional Relationships to Other DNA Polymerases" Proc. Natl. Acad. Sci. USA 86:4465-4469.
Abramson Richard D.
Gelfand David H.
Gould George M.
Hendricks Keith D.
Hoffmann-La Roche Inc.
Sias Stacey R.
Tramaloni Dennis P.
LandOfFree
5' to 3' exonuclease mutations of thermostable DNA polymerases does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with 5' to 3' exonuclease mutations of thermostable DNA polymerases, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and 5' to 3' exonuclease mutations of thermostable DNA polymerases will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1220375