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Cloned DNA polymerases from thermotoga and mutants thereof

a dna polymerase and thermotoga technology, applied in the field of cloned dna polymerases from thermotoga and mutants thereof, can solve the problem that relatively little investigation has been done to isolate and even clone these enzymes, and achieve the effect of reducing or eliminating the 5′-3′ exonuclease activity of the polymerase, reducing or eliminating discriminatory behavior

Inactive Publication Date: 2009-06-18
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides new DNA polymerases that are more thermostable than existing polymerases, with improved activity at high temperatures. These polymerases are isolated from the thermophilic bacteria Thermotoga maritima and Thermotoga neapolitana. The DNA polymerases of the invention are more stable than the commonly used polymerase Taq, with some versions showing no loss of activity even after heating for 60 minutes at 90°C with or without detergent. The DNA polymerases can be expressed in a variety of host cells, including prokaryotic and eukaryotic cells. Mutant versions of the polymerases are also provided that lack certain activities or are nondiscriminatory against certain nucleotides. The invention also includes methods for synthesizing double-stranded DNA molecules and sequencing them using the new polymerases.

Problems solved by technology

Although DNA polymerases from thermophiles are known, relatively little investigation has been done to isolate and even clone these enzymes.

Method used

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  • Cloned DNA polymerases from thermotoga and mutants thereof
  • Cloned DNA polymerases from thermotoga and mutants thereof
  • Cloned DNA polymerases from thermotoga and mutants thereof

Examples

Experimental program
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example 1

Bacterial Strains And Growth Conditions

[0151]Thermotoga neapolitana DSM No. 5068 was grown under anaerobic conditions as described in the DSM catalog (addition of resazurin, Na2S, and sulfur granules while sparging the media with nitrogen) at 85° C. in an oil bath from 12 to 24 hours. The cells were harvested by filtering the broth through Whatman #1 filter paper. The supernatant was collected in an ice bath and then centrifuged in a refrigerated centrifuge at 8,000 rpms for twenty minutes. The cell paste was stored at −70° C. prior to total genomic DNA isolation.

[0152]E. coli strains were grown in 2×LB broth base (Lennox L broth base: GIBCO / BRL) medium. Transformed cells were incubated in SOC (2% tryptone, 0.5% yeast extract, yeast 10 mM NaCl, 2.5 mM KCl, 20 mM glucose, 10 mM MgCl2, and 10 mM MgSO4 per liter) before plating. When appropriate antibiotic supplements were 20 mg / l tetracycline and 100 mg / l ampicillin. E. coli strain DH10B (Lorow et al., Focus 12:19-20 (1990)) was used ...

example 2

DNA Isolation

[0153]Thermotoga neapolitana chromosomal DNA was isolated from 1.1 g of cells by suspending the cells in 2.5 ml TNE (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM EDTA) and treated with 1% SDS for 10 minutes at 37° C. DNA was extracted with phenol by gently rocking the lysed cells overnight at 4° C. The next day, the lysed cells were extracted with chloroform:isoamyl alcohol. The resulting chromosomal DNA was further purified by centrifugation in a CsCl density gradient. Chromosomal DNA isolated from the density gradient was extracted three times with isopropanol and dialyzed overnight against a buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (TE).

example 3

Construction of Genomic Libraries

[0154]The chromosomal DNA isolated in Example 2 was used to construct a genomic library in the plasmid pCP13. Briefly, 10 tubes each containing 10 μg of Thermotoga neapolitana chromosomal DNA was digested with 0.01 to 10 units of Sau3Al for 1 hour at 37° C. A portion of the digested DNA was tested in an agarose (1.2%) gel to determine the extent of digestion. Samples with less than 50% digestion were pooled, ethanol precipitated and dissolved in TE. 6.5 μg of partially digested chromosomal DNA was ligated into 1.5 μg of pCP13 cosmid which had been digested with BamHI restriction endonuclease and dephosphorylated with calf intestinal alkaline phosphatase. Ligation of the partially digested Thermotoga DNA and BamHI cleaved pCP13 was carried out with T4 DNA ligase at 22° C. for 16 hours. After ligation, about 1 μg of ligated DNA was packaged using λ-packaging extract (obtained from Life Technologies, Inc., Gaithersburg, Md.). DH10B cells (Life Tech. Inc...

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Abstract

The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga (Tne and Tma) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3′→5′ exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5′→3′ exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant DNA polymerases in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The DNA polymerases of the invention may be used in well-known DNA sequencing and amplification reactions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This is a continuation-in-part of U.S. application Ser. No. 08 / ______, filed Aug. 14, 1996, pending, which is a continuation-in-part of U.S. application Ser. No. 08 / 537,400, filed Oct. 2, 1995, pending, which is a continuation-in-part of U.S. application Ser. No. 08 / 370,190, filed Jan. 9, 1995, pending, which is a continuation-in-part of U.S. application Ser. No. 08 / 316,423, filed Sep. 30, 1994, now abandoned. This is also a continuation-in-part of U.S. application Ser. No. 08 / 576,759, filed Dec. 21, 1995, which is a continuation of U.S. application Ser. No. 08 / 537,397, filed Oct. 2, 1995, which is a continuation-in-part of U.S. application Ser. No. 08 / 525,057, filed Sep. 8, 1995. The contents of each of these applications are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to a substantially pure thermostable DNA polymerase. Specifically, the DNA polymerase of...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N9/16C12N15/63C12N1/21C12P21/06C12P19/34C12N15/09C07H21/02C12N1/20C12N9/00C12N9/12C12N15/00C12N15/10C12N15/54C12N15/64C12R1/19
CPCC12N9/1252C12Q1/686C12Q1/6869C12Y207/07007C12Q2521/101
Inventor CHATTERJEE, DEB K.HUGHES, JR., A. JOHN
Owner LIFE TECH CORP
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