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US20110250598A1 - Detergent free polymerases - Google Patents

Detergent free polymerases Download PDF

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US20110250598A1
US20110250598A1 US13/042,628 US201113042628A US2011250598A1 US 20110250598 A1 US20110250598 A1 US 20110250598A1 US 201113042628 A US201113042628 A US 201113042628A US 2011250598 A1 US2011250598 A1 US 2011250598A1
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dna polymerase
detergent
thermostable dna
polymerase
chromatographic separation
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Ulrike Fischer
Michael Greif
Harald Sobek
Johann-Peter Thalhofer
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Roche Diagnostics Operations Inc
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Roche Diagnostics Operations Inc
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Publication of US20110250598A1 publication Critical patent/US20110250598A1/en
Priority to US14/160,453 priority Critical patent/US10227642B2/en
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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to the technical field of preparation and application of thermostable DNA polymerases. More precisely, the present invention relates provides a new method for production and use of thermostable DNA polymerases without any addition of detergent during production, storage or during application of the enzyme.
  • Thermostable DNA polymerases are enzymes which have been isolated and recombinantly expressed for a long time since the establishment of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the performance of PCR is known to be at least partially hampered by the presence of trace amounts various different reagents such as detergents.
  • the presence of detergents is known to be essential for many polymerase purification protocols and long term stabilization of enzymes in general and DNA polymerases in particular.
  • WO 08/077,017 discloses polymerase formulations with anionic and zwitter-ionic detergents instead of non ionic detergents.
  • the present invention provides a formulation of a thermostable DNA polymerase which is completely free of detergents. Such a formulation may be obtained, if the selected purification method does not require the addition of a detergent at any purification step.
  • the present invention also provides a kit comprising a thermostable DNA polymerase formulation which is completely free of detergents or a reaction mixture comprising a thermostable DNA polymerase which is completely free of detergents.
  • the present invention is directed to the use of the inventive polymerase formulations as disclosed above.
  • inventive polymerase formulations are predominantly advantageous, when they are used for the amplification of a target nucleic acid by means of a PCR, preferably real time PCR and most preferably real time PCR, wherein the amplification product is detected by at least one pair of FRET hybridization probes.
  • the present invention provides a method for preparation of a thermostable DNA polymerases, wherein all steps of preparation are executed in the absence of any detergent.
  • such a method may comprise the following steps:
  • thermostable DNA polymerase is recombinantly expressed in the form of a fusion protein which comprises a His tag. Then the preparation comprises the step of purifying said fusion protein using a nickel-loaded ion affinity column.
  • FIG. 1 Result of experiment as disclosed in Example 6.
  • Target DNA was used in various amounts and titrated from 300 ng (A) 30 ng (B). 3 ng (C), 0.3 ng (D) 0.03 ng (E) and no DNA (F).
  • the Taq DNA polymerase used was prepared without the addition of detergents.
  • FIG. 2 Result of experiment as disclosed in Example 6.
  • Target DNA was used in various amounts and titrated from 300 ng (A) 30 ng (B). 3 ng (C), 0.3 ng (D) 0.03 ng (E) and no DNA (F).
  • the Taq DNA polymerase used contained detergents (0.5% TWEEN 20, 0.5% NONIDET NP-40).
  • FIG. 3 Result of experiment as disclosed in Example 6.
  • Target DNA was used in various amounts and titrated from 300 ng (A) 30 ng (B). 3 ng (C), 0.3 ng (D) 0.03 ng (E) and no DNA (F).
  • the Taq DNA polymerase Roche Applied Science Cat. No: 11 146 165 001 was used for this experiment.
  • the present invention is originating from the theoretical hypothesis that for some the enzymatic activity of trace amounts of detergents might somehow affect the performance of thermostable DNA polymerases under at least some specific conditions. As it will be shown in the examples, the hypothesis could actually be tested to be true.
  • the present invention provides a formulation of a thermostable DNA polymerase which is completely free of detergents.
  • formulation of a thermostable DNA polymerase is being understood as any preparation of an at least partially purified thermostable DNA polymerase, which has been isolated from a cell lysate.
  • the lysate may be obtained from organisms which naturally contain said thermostable DNA polymerase, or preferably from recombinantly modified host cells, which express the gene encoding said thermostable DNA polymerase.
  • Thermostable DNA polymerases are thermostable enzymes which have originally been isolated and cloned from thermophilic bacteria. These enzymes catalize the template dependent primer extension by means of creating a phosphodiester bond between the free 3′ OH group of said primer and the alpha-phosphate moiety of a desoxynucleotide, whereas pyrophosphate is simultaneously generated as a side product.
  • said template is a DNA template.
  • the template may be RNA.
  • thermostable DNA dependent DNA polymerase originating from Thermos aquaticus. It possesses two enzymatic activities: a 5′-3′ polymerase activity and a double-strand specific 5′-3′ exonuclease activity, which provides the enzyme with strand displacement capability.
  • thermostable DNA polymerases can be formulated according to the present invention.
  • the thermostable DNA polymerase is selected from a group consisting of Aeropyrum pernix, Archaeoglobus fulgidus, Desulfurococcus sp. Tok., Methanobacterium thermoautotrophicum, Methanococcus sp.
  • Thermococcus species zilligii, barossii, fumicolans, gorgonarius , JDF-3, kodakaraensis KODI, litoralis , species 9 degrees North-7, species JDF-3, gorgonarius , TY), Thermoplasma acidophilum, Thermosipho africanus, Thermotoga sp.
  • thermoautotrophicum e.g., maritima, neapolitana
  • Thermus species e.g., aquaticus, brockianus, filiformis, flavus, lacteus, rubens, ruber, thermophilus , ZO5.
  • thermostable DNA polymerase is a DNA template dependent polymerase.
  • thermostable DNA polymerase has additional reverse transcriptase activity and may be used for RT-PCR.
  • Tth DNA polymerase from Thermus thermophilus (Roche Diagnostics Cat. No. 11 480 014 001).
  • thermostable DNA dependent DNA polymerases such as Taq DNA polymerase lack double strand dependent 3′-5′ exonuclease activity which is also known as proofreading activity. Yet, the scope of the present invention also includes other themostable enzymes which possess such a proofreading activity such as Pwo Polymerase (Roche Applied Science Cat. No. 04 743 750 001), Tgo Polymerase and Pfu Polymerase.
  • compositions according to the present invention may comprise blends of one or more of the polymerases mentioned above.
  • thermostable DNA polymerase also may be reversibly inactivated as a result of a chemical modification. More precisely, heat labile blocking groups are introduced into the Taq DNA polymerase which renders the enzyme inactive at room temperature (U.S. Pat. No. 5,773,258). These blocking groups are removed at high temperature during a pre-PCR step such that the enzyme is becoming activated.
  • heat labile modification for example can be obtained by coupling citraconic anhydride or aconitric anhydride to the lysine residues of the enzyme (U.S. Pat. No. 5,677,152).
  • Enzymes carrying such modifications are meanwhile commercially available as Amplitaq Gold (Moretti, T., et al., Biotechniques 25 (1998) 716-22) or FastStart DNA polymerase (Roche Applied Science Cat. No. 04 738 284 001).
  • thermostable DNA polymerase is either Taq DNA polymerase or delta 288 Taq DNA polymerase as disclosed in US 2005/0037412 or said delta 288 Taq DNA polymerase associated with an aptamer as disclosed in U.S. Pat. No. 6,020,130.
  • a formulation of a thermostable DNA polymerase which is completely free of detergents is obtained by a purification method which does not require the addition of a detergent at any purification step.
  • the formulation may comprise a buffer system and other non detergent supplements.
  • Such a formulation may comprise one, several or all of the following components: Tris-buffer, EDTA, DTT, salt and glycerol.
  • such a formulation may comprise on, several or all of the following amounts of components: 10 to 50 mM Tris/HCl pH 7.5, 0.05-0.2 mM EDTA, 0, 5-2 mM DTT, 50-200 mM potassium chloride, and 20-80% glycerol.
  • any of the inventive polymerases disclosed above may be a component of a kit.
  • a kit may comprise only said formulation and a reaction buffer in which a respective polymerization reaction can efficiently take place.
  • a kit in addition may comprise one or several desoxynucleoside-triphosphates such as dATP, dGTP, dCTP, and/or dTTP or derivatives or analogs thereof.
  • such a kit may comprise additional reagents for performing a primer extension reaction in general or a polymerase chain reaction (PCR) in particular.
  • a kit in addition may comprise at least one primer which is capable of binding a nucleic acid template that shall be amplified.
  • said kit may comprise one or several primer pairs, each designed to amplify a specific fragment of the template DNA.
  • the primer component may be a pool of oligonucleotides with an at least partially randomized sequence.
  • the kit according to the present invention may comprise additional reagents which enable the detection of the products generated by the thermostable DNA polymerase.
  • these reagents may enable the detection of a PCR amplification product by means of real time PCR.
  • Respective kits according to the present invention may thus additionally may comprise a double strand specific fluorescent DNA binding agent such as SYBR Green (Molecular Probes, Inc., Invitrogen Cat. No. 4304886) or LC480 Resolight dye (Roche Applied Science Cat. No. 04 909 640 001).
  • respective kits according to the present invention may comprise labeled hybridization probes such as TaqMan hydrolysis probes (U.S. Pat. No. 5,804,375) or Molecular Beacons (U.S. Pat. No. 5,118,801).
  • respective kits comprise at least on or several pairs of FRET hybridization probes (U.S. Pat. No. 6,174,670).
  • any of the inventive polymerase formulations may be part of a reaction mixture for performing a template dependent primer extension reaction in general and PCR amplification reaction in particular.
  • a reaction mixture which is free of any trace amounts of detergent comprises
  • such a reaction mixture is a PCR reaction mixture free of any detergent and comprises
  • such PCR reaction mixtures may additionally comprise any additional reagents which are enable the detection of the products generated by the thermostable DNA polymerase in real time.
  • Respective mixtures according to the present invention may thus additionally may comprise double strand specific fluorescent DNA binding agents, TaqMan hydrolysis probes, Molecular Beacons or FRET hybridization probes, which have already been disclosed above.
  • thermostable DNA polymerase formulations without any detergents are useful for any type of nucleic acid amplification reaction.
  • they may be used for random amplification such as a random priming reaction or whole genome amplification.
  • said inventive formulations are particularly useful for amplification of a specific target nucleic acid by means of performing a PCR reaction, which may be a real time PCR reaction.
  • PCR reaction may be monitored in real time.
  • sample analysis occurs concurrently with amplification in the same tube within the same instrument.
  • the formation of PCR products is monitored in each cycle of the PCR. It is usually measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction.
  • DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis.
  • the present invention is also directed to the use of a formulation of a thermostable DNA polymerase that is completely free of detergents for the amplification by means of PCR and in particular real time PCR.
  • double-stranded DNA specific dyes may be used, which upon excitation with an appropriate wavelength show enhanced fluorescence only if they are bound to double-stranded DNA.
  • only those dyes may be used which like SYBR Green I, for example, do not affect the efficiency of the PCR reaction.
  • fluorescence labeled hybridization probes which only emit fluorescence upon binding to its target nucleic acid can be used.
  • a single-stranded hybridization probe is labeled with two components.
  • the first component is excited with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer.
  • the hybridization probe binds to the target DNA and is degraded by the 5′-3′ exonuclease activity of the Taq DNA polymerase during the subsequent elongation phase.
  • the excited fluorescent component and the quencher are spatially separated from one another and thus a fluorescence emission of the first component can be measured.
  • TaqMan hydrolysis probe assays are disclosed in detail in U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,538,848, and U.S. Pat. No. 5,487,972.
  • TaqMan hybridization probes and reagent mixtures are disclosed in U.S. Pat. No. 5,804,375.
  • Molecular Beacon hybridization probes are labeled with a first component and with a quencher, the labels preferably being located at both ends of the probe.
  • both components are in spatial vicinity in solution.
  • After hybridization to the target nucleic acids both components are separated from one another such that after excitation with light of a suitable wavelength the fluorescence emission of the first component can be measured (U.S. Pat. No. 5,118,801).
  • thermostable DNA polymerase that is completely free of detergents is used for amplification of a target nucleic acid by means of a PCR, characterized in that said real time PCR is monitored in real time by means of FRET hybridization probes.
  • the FRET hybridization probe test format is useful for all kinds of homogenous hybridization assays (Matthews, J. A., and Kricka, L. J., Analytical Biochemistry 169 (1988) 1-25). It is characterized by two single-stranded hybridization probes which are used simultaneously and are complementary to adjacent sites of the same strand of the amplified target nucleic acid. Both probes are labeled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that a fluorescence emission of the second component can be measured when both hybridization probes bind to adjacent positions of the target molecule to be detected. Alternatively to monitoring the increase in fluorescence of the FRET acceptor component, it is also possible to monitor fluorescence decrease of the FRET donor component as a quantitative measurement of hybridization event.
  • the FRET hybridization probe format may be used in real time PCR, in order to detect the amplified target DNA.
  • the FRET hybridization probe format has been proven to be highly sensitive, exact and reliable (U.S. Pat. No. 6,174,670).
  • the FRET hybridization probe detection format can be “multiplexed”. More precisely, in one reaction vessel, multiple targets may become amplified with multiple pairs of amplification primers and detected with multiple hybridization probes. In this case, said multiple probes are labeled with different detectable fluorescent dyes in order to detect and discriminate the multiple targets which are supposed to be found in the sample.
  • fluorescein or fluorescein derivatives are used as a FRET donor moiety in combination with different FRET acceptor moieties such as Cy-5, LC-Red-640, or LC-red 705.
  • a typical example for an instrument capable of performing multiplex real time PCR is the Roche Diagnostics LIGHTCYCLER (Cat. No. 3 531 414 201). It is a fast PCR system enabling kinetic on-line
  • the optical system of the current LIGHTCYCLER version 2.0 being commercially available contains one light source, a blue light emitting diode (470 nm LED) and six detection channels.
  • a defined signal threshold is determined for all reactions to be analysed and the number of cycles Cp required to reach this threshold value is determined for the target nucleic acid as well as for the reference nucleic acids such as the standard or housekeeping gene.
  • the absolute or relative copy numbers of the target molecule can be determined on the basis of the Cp values obtained for the target nucleic acid and the reference nucleic acid.
  • the fluorescence emitted by a sample is separated by a set of dichroic mirrors and filters into different wavelengths that can be recorded in one of the six detection channels. Due to the fluorescent compounds which are available on the market, this allows detection of the double-stranded DNA-binding dye SYBR Green I, dual color detection with the TaqMan Probe format and 4-color detection with the Hybridization Probe (HybProbe) format. Details of the LIGHTCYCLER system are disclosed in WO 97/46707, WO 97/46712 and WO 97/46714.
  • the present invention provides a method for preparation of a thermostable DNA polymerases, characterized in that all steps of preparation are executed in the absence of any detergent.
  • any preparation method which can be successfully implemented for purification of a thermostable DNA polymerase without any addition of a detergent in each of the steps necessary can be applied.
  • such a purification method can comprise the steps of
  • the lysate is preferentially derived from a recombinant prokaryotic cell, such as E. coli , which is genetically modified to express the gene encoding the desired thermostable DNA polymerase in high yield.
  • a recombinant prokaryotic cell such as E. coli
  • the pellet may be frozen and in the sate, cells may be disrupted by physical methods such as sonication or, preferably by treatment with a French pressure cell.
  • a buffer may be added which already contains appropriate protease inhibitors such as, e.g., PMSF, Leupeptin and the like.
  • nucleic acids contained in the lysate may be removed, either enzymatically or preferably by means of precipitation, for example with Polymin P. Both, nucleic acid precipitates and protein precipitates may be removed by centrifugation.
  • the supernatant may then be subjected to a first chromatographic step, using an affinity chromatographic column, which is preferably functioning according to the principle of hydrophobic interaction chromatography.
  • the affinity matrix is a Phenyl-SEPHAROSE, such as Phenyl-SEPHAROSE CL-4B (GE Healthcare Bio-Sciences).
  • a second affinity chromatography may be performed using a second affinity matrix which is different from the first affinity matrix.
  • the sample may be purified over a Heparin SEPHAROSE column, containing Heparin SEPHAROSE CL-6B.
  • a third chromatographic step which is preferably purification by means of hydroxyapatite chromatography.
  • thermostable DNA polymerase comprise a so called Poly-His-tag.
  • Polyhistidine-tags are often used for affinity purification of Polyhistidine-tagged recombinant proteins expressed in prokaryotic and other expression systems.
  • the recombinant bacterial cells are harvested via centrifugation and the resulting cell pellet lysed, e.g., by physical means under conditions as disclosed above.
  • the crude extract contains the recombinant protein among several other proteins and nucleic acids originating from the bacterial host.
  • nucleic acids contained in the sample at this stage may be digested by DNAse I.
  • the mixture is then loaded onto a column comprising a specific affinity matrix such as nickel or cobalt loaded SEPHAROSE or the like.
  • a respective SEPHAROSE or agarose matrix contains bound nickel or cobalt ions to which the polyhistidine-tag binds with high affinity.
  • the resin is subsequently washed with a buffer to remove other proteins that do not specifically interact with the cobalt or nickel ion.
  • a second chromatographic step which is preferably an anion exchange chromatography.
  • a Q-SEPHAROSE ff column may be used.
  • a typical purification method for purifying his-tagged thermostable DNA polymerases may comprise the steps of
  • a dialysis step may be performed after each chromatographic elution, if required.
  • a dialysis is particularly advantageous in order to transfer the purified thermostable DNA polymerase into an appropriate storage buffer.
  • a suitable buffer system for long term storage at ⁇ 20° C. is 20 mM Tris/HCl, 0.1 mM EDTA, 100 mM potassium chloride, 1 mM DTT, 50% glycerol, pH 8.0.
  • Recombinant Taq DNA polymerase was purified to homogeneity from frozen E. coli cells K12LE392 harbouring the plasmids pUBS520 and pT5-Taq. Protein concentrations were measured at 280 nm. A molar extinction factor of 1.64 was used.
  • Frozen cells (25 gram) were thawed and suspended in 60 ml buffer A (50 mM Tris/HCl, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM EDTA, 0.64 ⁇ g/ml Leupeptin, pH 8.0). Cells were disrupted using a French pressure cell.
  • buffer A 50 mM Tris/HCl, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM EDTA, 0.64 ⁇ g/ml Leupeptin, pH 8.0.
  • the pool was applied on a Heparin SEPHAROSE column CL-6B (1.6 ⁇ 12.5 cm) equilibrated with buffer E (50 mM Tris/HCl, 0.1 mM EDTA, 100 mM KCl, 5% glycerol, pH 8.0). The column was washed using buffer E. The enzyme was eluted with a linear gradient of buffer E and buffer E+650 mM potassium chloride. Fractions were analyzed by SDS gel electrophoresis and the fractions containing the enzyme were pooled. The pooled fractions were dialyzed against buffer F (10 mM potassium phosphate, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, pH 8.0).
  • buffer F 10 mM potassium phosphate, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, pH 8.0.
  • the dialyzed pool was loaded on a HA Ultrogel (Pall, 1.6 ⁇ 8.5 cm). The column was washed with buffer F. The enzyme was eluted with a linear gradient of buffer F and buffer G (500 mM potassium phosphate, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, pH 8.0). The fractions containing the enzyme were pooled and dialyzed against detergent free storage buffer (20 mM Tris/HCl, 0.1 mM EDTA, 100 mM potassium chloride, 1 mM DTT, 50% glycerol, pH 8.0).
  • the recombinant truncated version of Taq DNA polymerase, delta 288 Taq DNA polymerase was purified to homogeneity from frozen E. coli K12XL1 blue cells harbouring the plasmid pQE80-L. Protein concentrations were measured at 280 nm. A molar extinction factor of 1.117 was used.
  • Frozen cells (20 gram) were thawed and suspended in 240 mL1 buffer A (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazol, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM DTT, pH 8.0). Cells were disrupted using a French pressure cell.
  • buffer A 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazol, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM DTT, pH 8.0.
  • MgCl 2 was added to a final concentration of 4 mM. After addition of DNase (50 u/ml) the solution was incubated at room temperature for 30 min. The solution was incubated at to 72° C. for 30 min. After cooling the solution to 2-8° C., the precipitated proteins were removed by centrifugation (10 min at 13 000 rpm).
  • the clear supernatant was applied to a nickel-loaded chelating SEPHAROSE ff column (5 ⁇ 4 cm) equilibrated in buffer B (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0).
  • buffer B 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0
  • the enzyme was eluted in with a linear gradient of buffer B and buffer C (50 mM sodium phoshate, 300 mM NaCl, 250 mM imidazole, pH 8.0). Fractions were analyzed by SDS gel electrophoresis and the fractions containing the enzyme were pooled.
  • the final pool was dialysed against detergent-free storage buffer (20 mM Tris/HCl, 0.1 mM EDTA, 1 mM DTT, 100 mM potassium chloride, 50% glycerol, pH 8.0).
  • the sequence of the aptamer was: (SEQ ID NO: 2) CGA TCA TCT CAG AAC ATT CTT AGC GTT TTG TTC TTG TGT ATG ATC G-PO 4
  • a highly concentrated enzyme solution was blended with aptamer and diluted with detergent-free storage buffer to final concentrations of 50 U/ ⁇ l polymerase and 4.33 pmol aptamer/unit of polymerase. Additionally, a version having a volume activity of 5 U/ ⁇ l was produced in the same way.
  • the enzyme blends were stored at ⁇ 20° C.
  • DNA polymerase activity was determined in a primer extension assay using standard procedures.
  • a primer/template hybrid was used as substrate.
  • the primer/template consisted of M13 sequencing primer 5′-GTA AAA CGA CGG CCA GT-3′ (SEQ ID No: 1) hybridized to M13 mp9ss DNA template.
  • the primer was extended by the incorporation of dNTPs.
  • the dNTPs mix contained radioactively labeled ⁇ 32 dCTP.
  • the synthesized product was precipitated with TCA and the incorporated ⁇ 32 dCTP was quantified using a scintillation counter.
  • Reactions were carried out in a 50 ⁇ l volume containing the following reagents: 67 mM Tris (pH 8.3), 5 mM MgCl 2 , 10 mM mercaptoethanol, 0.2% polydocanol, 0.2 mg/ml gelatine, 200 ⁇ M dATP, 100 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP, DNA/primer hybrid (1 ⁇ g DNA, 0.3 ⁇ g primer) and ⁇ 32 dCTP (1 ⁇ C). Aliquots of diluted enzyme were added to the mix, mixed and incubated for 60 min at 65° C. After incubation the samples were placed on ice and DNA was precipitated with 10% TCA solution. Samples were filtered through GFC-filters (Whatman), the filters were washed three times with 5% TCA, dried and counted in a ⁇ -counter in 2 ml scintillation fluid.
  • a fragment of the tPA gene was amplified from human genomic DNA (Roche Applied Science, Mat. No. 11 691 112). Detection and quantification of the amplified products was done using FRET hybridization probes (U.S. Pat. No. 6,174,670).
  • Formulation 3 delta 288 Taq DNA polymerase with detergent (0.5% TWEEN 20)
  • tPA7reverse GGA AGT ACA GCT CAG AGT TCT (SEQ ID NO: 4)
  • tPA7forward CTC CAT TCA TTC TCA AAA GGA CT
  • the detection oligonucleotides were:
  • tPA Fluos (SEQ ID NO: 5) GGG AAA GGC GGG GTG G-Fluo tPA LC-Red 640: (SEQ ID NO: 6) LC-Red 640-GCC ACT TAC CCT CAG AGC AGG CA
  • a low crossing point number corresponds to a high degree of amplified nucleic acid.
  • reaction ng template 300 30 3 0.3 0.03 Delta 288 Apta Taq without 21.5 24.9 28.2 31.6 35.2 detergent: Delta 288 Taq without detergent 22.8 26.7 29.5 30.6 32.5 Delta 288 with detergent 23.7 27.9 31.0 33.3 34.1

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EP2374874A3 (fr) 2012-01-11
US20140134633A1 (en) 2014-05-15
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