Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Nucleic acid encoding bacillus stearothermophilus tau polymerase subunit

a technology of bacillus stearothermophilus and polymerase, which is applied in the direction of enzymology, organic chemistry, transferases, etc., can solve the problems of lack of 3' to 5' exonuclease activity, limited thermostability of exant polymerases, and inability to obtain extended lengths of nucleotides

Inactive Publication Date: 2004-03-04
ODONNELL MICHAEL E +5
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is about thermostable DNA polymerases, specifically those derived from thermophilic bacteria, and their use as chromosomal replicases. These polymerases have been found to be more stable and effective in amplifying DNA compared to other polymerases. The invention includes the use of DNA polymerase III-type enzymes, which are composed of multiple subunits and have high processivity and rapid synthesis rates. The invention also includes the use of the entire holoenzyme particle, which consists of the DNA polymerase III core, the DNA polymerase III-related proteins, and the 5 protein .gamma. complex as a clamp loader. The invention provides a way to improve the efficiency and speed of DNA amplification and preparation of longer strands of DNA."

Problems solved by technology

Each of the references states that a drawback of the extant polymerases are their limited thermostability, and consequent useful life in the participation in PCR.
Such limitations also manifest themselves in the inability to obtain extended lengths of nucleotides, and in the instance of Taq polymerase, the lack of 3' to 5' exonuclease activity, and the drawback of the inability to excise misinserted nucleotides (Perrino, 1990).
However, previous work did not fully reconstitute the working replication machinery from fully recombinant subunits.
Mismatch incorporation during the synthesis or extension of the newly synthesized DNA molecule may result in one or a number of mismatched base pairs.
However, a thermostable DNA pol III-type complex used in these methods need only be added once at the start of the amplification (as for Taq DNA polymerase in traditional PCR amplifications), as its activity will be unaffected by the high temperature of the denaturation step.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nucleic acid encoding bacillus stearothermophilus tau polymerase subunit
  • Nucleic acid encoding bacillus stearothermophilus tau polymerase subunit
  • Nucleic acid encoding bacillus stearothermophilus tau polymerase subunit

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0330] Experimental Procedures

[0331] Materials

[0332] DNA modification enzymes were from New England Biolabs. Labelled nucleotides were from Amersham, and unlabeled nucleotides were from New England Biolabs The Alter-1 vector was from Promega. pET plasmids and E. coli strains, BL21 (DE3) and BL21(DE3)pLysS were from Novagen. Oligonucleotides were from Operon. Buffer A is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mMDTT, and 10% glycerol.

[0333] Genomic DNA

[0334] Thermus thermophilus (strain HB8) was obtained from the American Type Tissue Collection. Genomic DNA was prepared from cells grown in 0.1 l of Thermus medium N697 (ATCC: 4 g yeast extract, 8.0 g polypeptone (BBL; 11910), 2.0 g NaCl, 30.0 g agar, 1.0 L distilled water) at 75.degree. C. overnight. Cells were collected by centrifugation at 4.degree. C. and the cell pellet was resuspended in 25 ml of 100 mM Tris-HCl (pH 8.0), 0.05 M EDTA, 2 mg / ml lysozyme and incubated at room temperature for 10 min. Then 25 ml 0.10 M EDTA (pH 8.0), ...

example 2

[0364] Frameshifting Analysis of the T.th. dnaX Gene

[0365] Frameshifting was analyzed by inserting the frameshift site into lacZ in the three different reading frames, followed by plating on X-gal and scoring for blue or white colony formation (Weiss et al., 1987). The frameshifting region within T.th dnaX was subcloned into the EcoRI / BamHI sites of pUC19. These sites are within the polylinker inside of the .beta.-galactosidase gene. Three constructs were produced such that the insert was either in frame with the downstream coding sequence of .beta.-galactosidase, or were out of frame (either -1 or -2). An additional three constructs were designed by mutating the frameshift sequence and then placing this insert into the three reading frames of the .beta.-galactosidase gene. These six plasmids were constructed as described below.

[0366] The upstream primer for the shifty sequences was 5'-gcg cgg atc cgg agg gag aaa aaa aaa gcc tca gcc ca-3' (SEQ. ID. No. 10). The BamHI site for clonin...

example 3

[0369] Expression Vector for T.th. .gamma. and .tau.

[0370] The dnaX gene was cloned into the pET16 expression vector in the steps shown in FIG. 9. First, the bulk of the gene was cloned into pET16 by removing the PmlI / XbaI fragment from pAlterdnaX, and placing it into SmaI / XbaI digested Puc19 to yield Puc19dnaXCterm. The N-terminal sequence of the dnaX gene was then reconstructed to position an NdeI site at the N-terminus. This was performed by amplifying the 5' region encoding the N-terminal section of .gamma. / .tau. using an upstream primer containing an NdeI site that hybridizes to the dnaX gene at the initiating gtg codon (i.e. to encode Met where the Met is created by the PCR primer, and the Val is the initiating gtg start codon of dnaX). The primer sequence for this 5' end was: 5'-gtggtgcatatg gtg agc gcc ctc tac cgc c-3' (SEQ. ID. No. 15) (where the NdeI site is underlined, and the coding sequence of dnaX follows). The downstream primer hybridizes past the PmlI site at nucleot...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
temperatureaaaaaaaaaa
temperatureaaaaaaaaaa
temperatureaaaaaaaaaa
Login to View More

Abstract

The present invention relates to an isolated DNA molecule from a thermophilic bacterium which encodes a DNA polymerase III-type enzyme subunit. Also encompassed by the present invention are host cells and expression system including the heterologous DNA molecule of the present invention, as well as isolated replication enzyme subunits encoded by such DNA molecules. Also disclosed is a method of producing a recombinant thermostable DNA polymerase III-type enzyme, or subunit thereof, from a thermophilic bacterium, which is carried out by transforming a host cell with at least one heterologous DNA molecule of the present invention under conditions suitable for expression of the DNA polymerase III-type enzyme, or subunit thereof, and then isolating the DNA polymerase III-type enzyme, or subunit thereof.

Description

[0001] The present application is a continuation of U.S. patent application Ser. No. 09 / 716,964, filed Nov. 21, 2000, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 642,218, filed Aug. 18, 2000, as a continuation of U.S. patent application Ser. No. 09 / 057,416 filed Apr. 8, 1998, which claims the benefit of U.S. Provisional Patent Application Serial No. 60 / 043,202 filed Apr. 8, 1997, all of which are hereby incorporated by reference in their entirety.[0003] The present invention relates to thermostable DNA polymerases and, more particularly, to such polymerases as can serve as chromosomal replicases and are derived from thermophilic bacteria. More particularly, the invention extends to DNA polymerase III-type enzymes from thermophilic bacteria, including Aquifex aeolicus, Thermus thermophilus, Thermotoga maritima, and Bacillus stearothermophilus, as well as purified, recombinant or non-recombinant subunits thereof and their use, and to isolated DNA coding for ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/04C12N1/21C12N9/12C12Q1/68
CPCC07H21/04C12Q1/689C12N9/1252
Inventor O'DONNELL, MICHAEL E.YUZHAKOV, ALEXANDERYURIEVA, OLGAJERUZALMI, DAVIDBRUCK, IRINAKURIYAN, JOHN
Owner ODONNELL MICHAEL E
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com