WO1994014978A1 - Production amelioree de produits de la pcr en presence d'une proteine se fixant sur un seul brin de l'adn, et preparation de celle-ci a partir de thermus flavus - Google Patents

Production amelioree de produits de la pcr en presence d'une proteine se fixant sur un seul brin de l'adn, et preparation de celle-ci a partir de thermus flavus Download PDF

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Publication number
WO1994014978A1
WO1994014978A1 PCT/EP1993/003585 EP9303585W WO9414978A1 WO 1994014978 A1 WO1994014978 A1 WO 1994014978A1 EP 9303585 W EP9303585 W EP 9303585W WO 9414978 A1 WO9414978 A1 WO 9414978A1
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WIPO (PCT)
Prior art keywords
strand
organism
thermus flavus
dna
dna single
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PCT/EP1993/003585
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German (de)
English (en)
Inventor
Hans-B. Strack
Peter Kainz
Karim Othmann-Hassan
Original Assignee
Strack Hans B
Peter Kainz
Othmann Hassan Karim
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Strack Hans B, Peter Kainz, Othmann Hassan Karim filed Critical Strack Hans B
Publication of WO1994014978A1 publication Critical patent/WO1994014978A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • the polymerase chain reaction is a method of multiplying specific DNA sections (including genes) from any organism in a test tube with a rapidly expanding abundance of applications, especially in medical diagnostics, genetic engineering and basic biological research.
  • a suitable DNA polymerase in a suitable buffer, the four deoxiribonucleoside triphosphates as synthesis precursors and, in large excess, two generally chemically synthesized oligonucleotides are added.
  • These oligonucleotides must be sequence-complementary to one end or to the end of the opposite strand of the DNA section to be amplified.
  • the strands of the DNA are separated as single strands.
  • hybrids with the above-mentioned oligonucleotides are preferably formed, which thereby become primers (starting points) for the beginning of the synthesis and the respective addition to the double strand.
  • the (ideally) doubling after another denaturation is followed by a further doubling cycle and the process is repeated until a limiting factor occurs, e.g. the exhaustion of the synthesis precursors, the polymerase or the self-restoration of the synthesis products.
  • Suitable polymerases in the abovementioned sense are only suitable for commercial applications and for scientific research tasks if they have been isolated from or derived from theimophilic organisms - and only because this is the only way to carry out the repeated denaturation steps of the process without that each time anew polymerase must be added to the batch - again a crucial prerequisite for the automation of the process.
  • the specificity of the method is i. a. satisfactory, d. H. only the DNA segments selected by specifying the oligonucleotides used are propagated.
  • the present invention consists in isolating or enriching DNA single-strand-binding proteins from thermophilic organisms and adding these proteins isolated in this way or proteins genetically derived from them or the preparations enriched on them in batches of the polymerase chain reaction.
  • the approach carried out using the single-strand-binding protein results in a considerable increase in the yield of the polymer chain reaction in comparison to the control (lane 1) and thus at least the possibility of using to achieve an equivalent result in one fifth of the use of polymerase. It is shown using an example that a single-strand binding protein from a thermophilic organism, in the present case Thermus flavus, can interact with a polymerase from a thermophilic organism, in the present case also Thermus flavus, and this for improvement the polymer chain reaction can be used.
  • Fig. 1 shows the result of the gel retardation assay with the protein concentrate which was isolated according to Example 1, lanes 2, 6 and 7 belonging to approaches in which no concentrate was used.
  • Fig. 2 shows the result of a polymerase chain reaction (hereinafter also abbreviated as PCR) with and without the addition of the protein concentrate from Example 1, lane 1 relating to the reaction batch without addition and lane 2 relating to the reaction batch with addition.
  • PCR polymerase chain reaction
  • Lane 3 is a so-called kilobase ladder, ie a calibration standard for determining molecular weight double-stranded DNA (Bethesda Research Laboratories,
  • FIG. 3 shows the optical density according to the densitometric evaluation (negative film) of lane 1 of Fig. 2.
  • Fig. 4 shows the optical density of the web 2 of Fig. 2 and
  • Fig. 5 shows the optical density of the web 3 of Fig. 2.
  • thermophilic bacterium Thermus flavus Process for isolating a DNA single-strand-binding protein from the thermophilic bacterium Thermus flavus.
  • Bacteria from the strain Thermus flavus - (German collection of microorganisms., Braunschweig, strain AT-62, No. 674) were in the culture medium DSM No. 74 (8 g polypeptone, 4 g yeast extract, 2 g sodium chloride / liter; pH 7.0) under the following Conditions drawn up: overnight at 75 ° C. in a shaking incubator (New Brunswick, 15Orpm) to an optical density of 1.56, measured at 600 nm. The bacterial cells were cooled to room temperature, collected as sediment by centrifugation and, until further use, at - Stored at 70 ° C. The yield was 1.2 g per liter (wet weight).
  • the pipetted supernatant was brought to a final concentration of 0.2M with potassium phosphate pH 6.5 and applied to a chromatography column filled with DEAE cellulose (DE 52 cellulose, Whatman; bed volume 200 ml).
  • DEAE cellulose was previously equilibrated with 0.2M potassium phosphate pH 6.5, 10mM beta-mercaptoethanol (buffer A). Unbound proteins were eluted with BufferA. The elution volume was 100 ml.
  • the eluate from the above chromatography (containing the unbound proteins) was mixed with 4 parts of 10 mM beta-mercaptoethanol and applied to another DEAE cellulose chromatography column as described above, but with a bed volume of only 40 ml.
  • the column was rinsed with 20 ml of 20 mM potassium phosphate pH 6.5, 10 mM beta-mercaptoethanol (buffer B) and the bound proteins were eluted using a linear gradient of buffer B and - 200 mM potassium phosphate, 10 mM beta-mercaptoethanol.
  • the elution volume was 100 ml, the volume of the individual fractions 5 ml.
  • the elution volume was 60 ml, the volume of the individual fractions 5 ml.
  • the fractions containing the approx. 13 kd protein were in a concentration range between 200 and 220 mM phosphate. These were combined and triple volume 0.1 mM EDTA (ethylenediaminetetraacetic acid) pH 8.2, 2 mM DTE (dithioerythrol), 10% glycerol (v / v) was added.
  • the combined and diluted fractions from the phosphocellulose chromatography were further fractionated by means of heparin-Sepharose chromatography (Pharmacia).
  • the approximately 13 kd protein was eluted in the fractions in the range between 350 and 400 mM KCI. As with the above fractionation steps, this was verified by SDS polyacrylamide gel electrophoresis. The corresponding fractions were combined, in a final concentration step mixed with twice the volume of buffer C, applied to a heparin-Sepharose column (bed volume: 1 ml) and eluted with 0.6M KCI in buffer C. The volume of the eluate was 400 ⁇ l. An analysis of an aliquot of this protein fraction by means of SDS polyacrylamide gel electrophoresis showed that the main protein fraction (> 90%) consisted of the ca13kd protein.
  • 13kd protein Designated concentrate This eluate was dialyzed overnight at 4 ° C against Tub Polymerase Storage Buffer (Amersham Cat.No.T0333Z), mixed with glycerin (final conc. 50%) and stored at -20 ° C. It is hereinafter referred to as 13kd protein Designated concentrate.
  • 13kd protein Designated concentrate By means of a gel retardation assay (see below) and single-stranded DNA cellulose chromatography, it was shown that the isolated 13kd protein is a single-stranded DNA, regardless of its nucleotide sequence binding protein.
  • the 13kd protein concentrate was prepared with both double-stranded and three single-stranded DNA oligonucleotides of different sequences.
  • the batches were incubated at 70 ° C. for 30 minutes and then analyzed by means of polyacrylamide gel electrophoresis (4-20% TB gradient gels, NOVEX CHEM 125 V, 50 minutes).
  • the nucleic acids were detected by means of ethidium bromide fluorescence.
  • Lanes 1 to 6 are from one electrophoresis run, lanes 7 to 9 are from another.
  • the result of the electrophoresis shows that the DNA-protein binding of the purified 13 kd protein was carried out exclusively to single-stranded DNA in a sequence-unspecific manner.
  • Deoxynucleoside triphosphates as 100mM stock solutions, pH 7.5; Ultrapure,
  • Buffer Tris ultrapur, BRL; Ammonium sulfate, Merck, MgCl 2, Merck
  • the 50 ⁇ l reaction mixtures contained 1 ⁇ M of each primer, 0.2mM of each dNTP, 10pg ⁇ -DNA, 5 ⁇ l 10x PCR buffer and 0.25 U hot tub polymerase (Fig. 2, lane 1) or 0.25U polymerase + 2 ⁇ l des purified 13kd protein. (Fig. 2, lane 2)
  • the amplification was carried out in two steps in the form of a 2-step PCR as follows:
  • cycle No. 1 The initial denaturation of the DNA (cycle No. 1) was carried out at 94 ° C. for 4 min.
  • Cycles 2-11 consisted of 65 ° C for 1.5 min (annealing of the primers and extension) and 94 ° C for 4 sec (denaturation step: separation of the complementary strands as a prerequisite for annealing new primers in the next PCR cycle).
  • the final primer annealing and extension step (cycle # 12) was at 65 ° C for 3 min.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Le déroulement de réactions en chaîne de la polymérase est amélioré du fait qu'on ajoute au produit initial de la réaction des préparations de protéines se fixant sur un seul brin de l'ADN, provenant d'organismes thermophiles. Les préparations appropriées peuvent être obtenues à partir d'organismes thermophiles, notamment du Thermus flavus, souche AT-62, DSM no. 674, par fractionnement d'un extrait brut, obtenu à partir de cellules d'un tel organisme.
PCT/EP1993/003585 1992-12-18 1993-12-16 Production amelioree de produits de la pcr en presence d'une proteine se fixant sur un seul brin de l'adn, et preparation de celle-ci a partir de thermus flavus WO1994014978A1 (fr)

Applications Claiming Priority (2)

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AT251292 1992-12-18
ATA2512/92 1992-12-18

Publications (1)

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WO1994014978A1 true WO1994014978A1 (fr) 1994-07-07

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039525A1 (fr) * 1995-06-06 1996-12-12 The Mount Sinai Medical Center Of The City University Of New York Clonage et expression de mutants thermostables de genes et de proteines, et leurs utilisations
EP1985703A2 (fr) 1994-11-02 2008-10-29 Allelix Neuroscience, Inc. Système nerveux périphérique à canaux de sodium spécifiques, son codage ADN, cristallisation, diffraction de rayon X, modélisation moléculaire informatique, concept de médicament rationnel, criblage de drogue, et ses procédés de fabrication et d'utilisation
US7662593B2 (en) 2004-12-21 2010-02-16 Aisin Seiki Kabushiki Kaisha Activation method of protein derived from extremely thermophilic bacterium in nucleic acid amplification reaction and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0395398A2 (fr) * 1989-04-27 1990-10-31 Life Technologies Inc. Amplifications de séquences d'acide nucléique utilisant des séquences oligonucléotiques au hasard comme amorces
WO1991006679A1 (fr) * 1989-10-24 1991-05-16 Stratagene Procede ameliore d'hybridation des acides nucleiques utilisant une proteine monocatenaire liante de l'acide nucleique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0395398A2 (fr) * 1989-04-27 1990-10-31 Life Technologies Inc. Amplifications de séquences d'acide nucléique utilisant des séquences oligonucléotiques au hasard comme amorces
WO1991006679A1 (fr) * 1989-10-24 1991-05-16 Stratagene Procede ameliore d'hybridation des acides nucleiques utilisant une proteine monocatenaire liante de l'acide nucleique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J.K. BALL AND U. DESSELBERGER: "Incorporation of single-stranded DNA binding protein early in polymerase chain reaction product sequencing reactions prevents enzyme pausing", ANALYTICAL BIOCHEMISTRY, vol. 207, no. 2, December 1992 (1992-12-01), ACADEMIC PRESS, INC., NEW YORK, US;, pages 349 - 351 *
K. SCHWARZ ET AL.: "Improved yields of long PCR products using gene 32 protein", NUCL. ACIDS RES., vol. 18, no. 4, 25 February 1990 (1990-02-25), IRL PRESS, OXFORD, ENGLAND;, pages 1079 *
M. PANACCIO AND A. LEW: "PCR based diagnosis in the presence of 8% (v/v) blood", NUCL. ACIDS RES., vol. 19, no. 5, 11 March 1991 (1991-03-11), IRL PRESS, OXFORD, ENGLAND;, pages 1151 *
R.G. OSHIMA: "Single-stranded DNA binding protein facilitates amplification of genomic sequences by PCR", BIOTECHNIQUES, vol. 13, no. 2, August 1992 (1992-08-01), EATON PUBL. CO., MA,US;, pages 188 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1985703A2 (fr) 1994-11-02 2008-10-29 Allelix Neuroscience, Inc. Système nerveux périphérique à canaux de sodium spécifiques, son codage ADN, cristallisation, diffraction de rayon X, modélisation moléculaire informatique, concept de médicament rationnel, criblage de drogue, et ses procédés de fabrication et d'utilisation
WO1996039525A1 (fr) * 1995-06-06 1996-12-12 The Mount Sinai Medical Center Of The City University Of New York Clonage et expression de mutants thermostables de genes et de proteines, et leurs utilisations
US5877280A (en) * 1995-06-06 1999-03-02 The Mount Sinai School Of Medicine Of The City University Of New York Thermostable muts proteins
US7662593B2 (en) 2004-12-21 2010-02-16 Aisin Seiki Kabushiki Kaisha Activation method of protein derived from extremely thermophilic bacterium in nucleic acid amplification reaction and use thereof

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