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WO2000071739A9 - Activite de transcription inverse de l'adn polymerase issu de bacillus stearothermophilus, en presence de magnesium - Google Patents

Activite de transcription inverse de l'adn polymerase issu de bacillus stearothermophilus, en presence de magnesium

Info

Publication number
WO2000071739A9
WO2000071739A9 PCT/US2000/013960 US0013960W WO0071739A9 WO 2000071739 A9 WO2000071739 A9 WO 2000071739A9 US 0013960 W US0013960 W US 0013960W WO 0071739 A9 WO0071739 A9 WO 0071739A9
Authority
WO
WIPO (PCT)
Prior art keywords
polymerase
magnesium
rna
dna
dna polymerase
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2000/013960
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English (en)
Other versions
WO2000071739A1 (fr
Inventor
Judith E T Schanke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Epicentre Technologies Corp
Original Assignee
Epicentre Technologies Corp
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.)
Filing date
Publication date
Application filed by Epicentre Technologies Corp filed Critical Epicentre Technologies Corp
Priority to EP00932671A priority Critical patent/EP1185680A4/fr
Priority to CA002374494A priority patent/CA2374494A1/fr
Priority to JP2000620116A priority patent/JP2003500063A/ja
Priority to AU50361/00A priority patent/AU765313B2/en
Publication of WO2000071739A1 publication Critical patent/WO2000071739A1/fr
Anticipated expiration legal-status Critical
Publication of WO2000071739A9 publication Critical patent/WO2000071739A9/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-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)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Definitions

  • the present invention is in the fields of molecular and cellular biology.
  • the invention is in the fields of molecular and cellular biology.
  • thermostable reverse transcriptase derived from Bacillus
  • the invention relates to methods for producing nucleic acid molecules (particularly
  • thermostable protein fragment having reverse transcriptase activity in
  • thermostable DNA polymerase enzymes combinations of reverse transcriptase and thermostable DNA polymerase enzymes using a
  • thermostable reverse transcriptase derived from Bacillus stearothermophilus.
  • reverse transcriptase describes a class of polymerases characterized as RNA-
  • Reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Reverse transcriptase has been used primarily to
  • RNA amplification methods such as RT-PCR, NASBA, TMA, 3SR, or SPSR.
  • Reverse transcription is commonly performed with viral reverse transcriptases isolated
  • AMV-RT Avian myeloblastosis virus
  • MMLV-RT Moloney murine leukemia virus
  • RNA denaturation step which can be
  • polymerases with reverse transcriptase activities are commonly isolated from Thermus species.
  • the polymerase copies the template RNA with low fidelity and the RNA
  • Reverse transcriptases have been extensively used in reverse transcribing RNA prior to
  • PCR amplification This method, often referred to as RT-PCR, is widely used for detection and
  • RNA template is first copied into cDNA using a reverse transcriptase
  • transcriptase a reaction termed "first-strand synthesis.” PCR is then performed to exponentially
  • the RT-PCR method entails three steps, namely:
  • first cDNA strand i.e., the primer must be identical or sufficiently homologous to a sequence
  • the reverse transcriptase activity is a component of the thermostable Tth DNA
  • Annealing and cDNA synthesis are performed in the presence of manganese ions
  • PCR is carried out in the presence of magnesium after the removal of manganese by a
  • RT-PCR method (“one-step") integrates the three RT-PCR steps into a single continuous reaction
  • thermostable DNA polymerase Tth thermostable DNA polymerase
  • RNA denaturation step was omitted.
  • thermostable DNA polymerase such as Tth
  • the polymerase copies the template RNA with low fidelity.
  • transcriptases such as AMV-RT and MMLV-RT, which are not dependent upon manganese for
  • RNA Reverse transcriptases Transcription-based Amplification of RNA Reverse transcriptases also have been used to reverse transcribe RNA during
  • transcription-based amplification techniques where these techniques may be classified either as
  • TAS transcription-based amplification system
  • the first stage makes it possible to synthesize a cDNA from RNA in the presence
  • the single-stranded cDNA is replicated by reverse transcriptase in the presence of
  • stage contains a double-stranded promoter to which a phage DNA-dependent RNA polymerase
  • the third stage then consists of transcribing RNA molecules (from 30 to 1000 per
  • NASBA Nucleic Acid Sequence-Based Amplification
  • TMA Transcription Mediated Amplification
  • SPSR Single Primer Sequence Replication
  • DNA-dependent DNA polymerase a retrovirus reverse transcriptase such as AMN-RT or a retrovirus reverse transcriptase
  • MMLV-RT ribonuclease H (R ⁇ ase H)
  • D ⁇ A-dependent R ⁇ A polymerase e.g., T7 bacteriophage R ⁇ A
  • R ⁇ A polymerase e.g., T7 phage binding site is introduced into the cD ⁇ A by the primer used
  • the free cD ⁇ A is then replicated from a second oligonucleotide primer by reverse
  • the D ⁇ A/D ⁇ A homoduplex is transcribed into R ⁇ A by T7 R ⁇ A polymerase and
  • this R ⁇ A can again serve as template for the next cycle.
  • reaction temperatures i.e., > 45 °C. Therefore, the reaction temperatures cannot be raised to prevent, for
  • thermostable reverse transcriptase that is active in the presence of magnesium ions
  • amplification methods such as RT-PCR, NASB A, TMA, 3 SR, or SPSR.
  • the present invention is generally directed to thermostable DNA polymerases from
  • Bacillus stearothermophilus which are mutated or truncated forms of the native enzyme
  • polymerase has a molecular mass of about 55 to 65 kDA as determined by SDS gel
  • molecules is at least 1 mM, and more preferably about 1.0 mM to about 10.0 mM, about 1.0 mM
  • a buffer or a magnesium-containing salt which may be magnesium chloride
  • magnesium sulfate or magnesium acetate, as well as other magnesium-containing buffers and
  • the invention is directed to methods for amplifying a nucleic acid molecule
  • RNA template comprising (a) mixing an RNA template with a composition comprising a truncated form of Bst
  • DNA polymerase sold as ISOTHERMTM DNA polymerase, Epicentre Technologies
  • the DNA sequence is complementary to all or a portion of the RNA template.
  • the DNA sequence is complementary to all or a portion of the RNA template.
  • thermostable DNA polymerases used are thermostable DNA polymerases, and most preferably Tne, Tma, Taq, Pfu,
  • Etb Pwo, Tfl, or a mutant, variant or derivative thereof.
  • the DNA polymerases may comprise a first
  • DNA polymerase having 3' exonuclease activity most preferably a DNA polymerase selected
  • a DNA polymerase selected from the group consisting of Taq, Tfl, Tth, and mutants,
  • the invention also is directed to such methods wherein the mixture further comprises one
  • nucleotides preferably deoxyribonucleoside triphosphates (most preferably dATP,
  • dUTP dideoxyribonucleoside triphosphates (most preferably ddATP,
  • nucleotides may optionally be
  • detectably labeled e.g. , with a radioactive or non-radioactive detectable label.
  • oligonucleotide primers which are preferably oligo(dT) primers, random primers,
  • primers or target-specific primers which is more preferably a gene-specific primer.
  • the invention also is directed to such methods wherein the incubating step comprises (a)
  • RNA template at a temperature and for a time sufficient to amplify the DNA molecule
  • thermocycling preferably via thermocycling, more preferably thermocycling comprising alternating heating and
  • thermocycling comprising alternating from a first temperature range of from about 90 ° C to about
  • thermocycling is performed greater than 20 times, more preferably greater than 30 times.
  • the present invention is directed to methods for amplifying a nucleic acid
  • transcription-based amplification techniques that include, but are not limited
  • NASBA to, NASBA, TMA, 3SR, or SPSR.
  • a part of the invention includes test kits for carrying out the previously described methods.
  • Figure 1 is a photograph of an ethidium bromide stained gel demonstrating the ability
  • Lane M contains a DNA sizing ladder. Lanes 1 and
  • Lanes 3 and 4 contain the
  • Figure 2 is a photograph of an ethidium bromide stained gel demonstrating the effect of
  • Lane M contains a 100 bp DNA sizing ladder; Lanes 1-4 contain 0, 1.0, 2.0,
  • Figure 3 is a photograph of an ethidium bromide stained gel demonstrating coupled
  • the 463 bp amplification product of a region of the tobacco mosaic virus (TMV) RNA is indicated with an
  • Lane M contains 100 bp DNA sizing ladder; Lane 1 contains the reaction with no
  • Figure 4 is a photograph of an ethidium bromide stained gel demonstrating a long RNA
  • thermostable DNA polymerases coupled with a mix of thermostable DNA polymerases.
  • Lane M contains
  • Lanes 1 and 2 are the products from reactions containing OX and IX
  • the present invention is directed to a thermostable and enzymatically active truncated
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • compositions comprising Bst enzymes having reverse transcriptase
  • compositions may be used in the methods of the invention to produce, analyze, quantitate and otherwise manipulate nucleic acid molecules using a one- or two-step
  • the present invention relates to a thermostable and enzymatically active truncated
  • protein may be purified according to any number of protocols known in the art (see e.g. , Ye, SN.
  • the purified full-length Bst DNA polymerase (167 ⁇ g/ml) (microgram/ml) in a standard
  • subtilisin (microgram/milliliter) subtilisin for 16 hours at room temperature.
  • the proteolysis reaction was
  • fractions were assayed ⁇ o ⁇ Bst polymerase activity and for purity by 10% SDS electrophoresis before pooling. The pool was dialyzed against the
  • fragment lacks 5'-3' exonuclease activity, and (3) that the fragment has reverse transcriptase
  • Particularly prefened enzymes for use in the invention include Rst reverse transcriptases,
  • the buffer in the compositions of the invention provide appropriate pH and ionic
  • nucleotides used in the compositions e.g., deoxyribonucleoside triphosphates (dNTPs)
  • dNTPs deoxyribonucleoside triphosphates
  • the primer nucleic acid molecules provide the substrates for synthesis or amplification of nucleic
  • compositions of the invention provide a concentration of magnesium-containing
  • molecules of at least 1 mM and more preferably about 1.0 mM to about 10.0 mM, about 1.0 mM
  • the source of the magnesium-containing molecules is a buffer or a magnesium-containing salt which may be magnesium chloride,
  • magnesium sulfate or magnesium acetate, as well as other magnesium-containing buffers and
  • compositions of the invention also comprise one or more DNA polymerases, which
  • thermostable DNA polymerases are preferably thermostable DNA polymerases. These DNA polymerases may be isolated from
  • thermophilic bacteria that are available commercially, or may be obtained by recombinant DNA
  • thermostable polymerases Suitable for use as sources of thermostable polymerases or the genes thereof for
  • thermophilic bacteria Thermus aquaticus, Thermus
  • thermophilus Thermococcus litoralis, Pyrococcusfuriosus, Pyrococcus wosei and other species
  • Thermotoga maritima and other species of the Thermotoga genus
  • thermoautorophicum Methanobacterium thermoautorophicum, and mutants variants or derivatives thereof. It is to be
  • thermostable DNA polymerases from other organisms may also be thermostable DNA polymerases from other organisms.
  • Thermostable DNA polymerases such as Taq is preferably added to the present
  • compositions at a final concentration in solution of about 25-100 units per milliliter, most
  • Bst large fragment (sold as ISOTHERM DNA polymerase,
  • the concentration of DNA polymerases is determined as a ratio of the concentration of the enzymes having reverse transcriptase activity.
  • reverse transcriptase activity to Taq DNA polymerase ranges from about 0.25:1 to about 16:1,
  • transcriptase enzymes to DNA polymerases suitable for use in the invention will be apparent to
  • compositions of the invention further comprise one or more nucleotides (e.g., one or more nucleotides),
  • dNTPs deoxyribonucleoside triphosphates
  • compositions serve as the building blocks for newly synthesized nucleic acids, being
  • nucleotides suitable for use in the present compositions include, but are not limited to, dUTP,
  • thio-dCTP or derivatives thereof, all of which are available commercially from various suppliers.
  • the dNTPs may be unlabeled, or they may be detectably labeled by coupling them by methods
  • Labeled dNTPs may also be obtained from commercial suppliers.
  • compositions, the dNTPs are added to give a working concentration of each dNTP of about
  • compositions comprise one or more primers which are
  • RNA template a first strand DNA molecule complementary (single-stranded cDNA molecule) to all or a portion of an RNA template.
  • primers may also be used to synthesize
  • these primers may be used in
  • Such primers include, but are not limited to,
  • target-specific primers which are preferably gene-specific primers
  • oligo(dT) are not limited to, target-specific primers (which are preferably gene-specific primers), oligo(dT)
  • primers random primers or arbitrary primers. Additional primers that may be used for
  • reaction mixtures are incubated at a temperature sufficient to produce a sample.
  • conditions typically occur at temperatures of at least 40 °C, and more preferably range from at
  • amplification is accomplished via one or more polymerase chain reactions (PCRs).
  • PCRs polymerase chain reactions
  • thermocycling which may comprise alternating heating
  • second temperature range of from about 45 °C to about 75 °C, preferably from about 60 °C to
  • thermocycling may be performed any number of
  • compositions and methods of the present invention may also be used for the
  • RT-PCR preferably nucleic acid molecules that are larger than about 3-6 kilobases in size
  • nucleic acid more preferably larger than about 4-5 kilobases in size, and most preferably nucleic acid
  • DNA polymerases preferably mixtures of one or more DNA polymerases
  • compositions of the invention Barnes, W.M., Proc.
  • thermostable polymerases Particularly prefened 3'-exo- polymerases
  • 3'-exo+ polymerases include,
  • thermostable reverse transcriptase i.e., AMN-RT or MMLV-RT
  • thermostable reverse transcriptase i.e., AMN-RT or MMLV-RT
  • thermostable R ⁇ ase H as described in U.S. Pat. Nos. 5,268,289 and 5,459,055 and 5,500,370, all of which are incorporated herein by
  • RNA polymerase e.g., T7 bacteriophage RNA polymerase
  • polymerase e.g., mutated forms from T3, T7, or SP6 RNA polymerases
  • thermostable conditions i.e., from about 40°C to about 80°C, wherein each mutant RNA
  • thermophilic phage RNA polymerase employs its own specific promoters; or (2) a thermophilic phage RNA polymerase,
  • thermostable RNA polymerase from native or recombinant sources, that itself encodes for a thermostable RNA polymerase
  • thermophilic organism e.g., Tth or Bst
  • thermophilic organism promoters from the respective thermophilic organism.
  • Example 1 Detection of reverse transcriptase activity in presence of magnesium ions
  • CCCTTTGCGGACATCACTCTT 3' (SEQ ID NO: 2), 500 ng of TMV RNA, and 20 U of Bst
  • One reaction contained no MgCl 2 , one contained 1.5 mM MgCl 2 , and one
  • primers having the following sequences: (forward) (5' GCCGGTTTGGTCGTCACGGGC 3')
  • a region of the rabbit tissue factor (RTF) mRNA transcript was used as a template for
  • the RTF-RNA transcript was generated by subcloning an RT-PCR
  • amplified rabbit brain mRNA product into a transcription vector containing a T7 promoter.
  • forward and reverse RTF primers used for the amplification had the following sequence:
  • Amplification conditions were as follows: reverse transcription was performed at 42 °C for 30
  • the RT-PCR product was ligated into a plasmid by standard methods.
  • RNA template was quantified by specfrophotometry. Two hundred and fifty picograms of
  • RNA transcript were used in the subsequent RT-PCR amplification reactions.
  • Reactions contained 50 mM Tris-HCl, (pH 9.0), 20 mM (NH 4 ) 2 SO 4 , 12.5 mM NaCl, 200 ⁇ M
  • One of the reactions also contained 0.5 mM MnSO 4 .
  • the reagents were incubated
  • the number of mutations per base sequenced was .0023 without manganese and was 0.0085 in
  • thermostable DNA polymerases combined with one or more thermostable DNA polymerases.
  • TMV tobacco mosaic virus
  • TCGCTTTATTACGTGCCTGC 3' (SEQ ID NO: 9), 200 ng of TMV RNA, 20 U Bst DNA 13.
  • said incubating step (b) comprises
  • a method for amplifying a nucleic acid molecule comprising
  • RNA template complementary to all or a portion of said RNA template.
  • thermocycling
  • thermocycling comprises alternating
  • thermocycling comprises alternating
  • thermocycling is performed greater than 5
  • thermostable DNA polymerases are
  • Tne selected from the group consisting of Tne, Tma, Taq, Pfu, Tth, Pwo, Tfl, and mutants, variants
  • DNA polymerase having 3' exonuclease activity and a second DNA polymerase having
  • activity is selected from the group consisting of Pfu, Pwo, Tne, Tma, and mutants, variants
  • reduced 3' exonuclease activity is selected from the group consisting of Taq, Tfl, Tth, and
  • DNA polymerases or claim 2 to said DNA polymerases is from about 0.25:1 to about 16:1.
  • nucleotides are deoxyribonucleoside
  • -29- selected from the group consisting of dATP, dUTP, dTTP, dGTP, and dCTP.
  • kits for synthesizing or amplifying a DNA molecule comprising one or more
  • the present invention is directed to a thermostable DNA polymerase from Bacillus
  • RT-PCR DNA chain reaction

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  • Health & Medical Sciences (AREA)
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  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne l'ADN polymérase thermostable issu de Bacillus Stearothermophilus et destiné à être utilisé dans une transcription inverse et/ou une transcription inverse suivie de réaction en chaîne de la polymérase (RT-PCR), dans laquelle l'ADN polymérase présente une activité de transcription inverse en présence d'ions magnésium et en l'absence substantielle d'ions manganeux.
PCT/US2000/013960 1999-05-22 2000-05-19 Activite de transcription inverse de l'adn polymerase issu de bacillus stearothermophilus, en presence de magnesium Ceased WO2000071739A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00932671A EP1185680A4 (fr) 1999-05-22 2000-05-19 Activite de transcription inverse de l'adn polymerase issu de bacillus stearothermophilus , en presence de magnesium
CA002374494A CA2374494A1 (fr) 1999-05-22 2000-05-19 Activite de transcription inverse de l'adn polymerase issu de bacillus stearothermophilus, en presence de magnesium
JP2000620116A JP2003500063A (ja) 1999-05-22 2000-05-19 マグネシウム存在下におけるバチルスステアロテルモフィルス由来ｄｎａポリメラーゼの逆転写活性
AU50361/00A AU765313B2 (en) 1999-05-22 2000-05-19 Reverse transcription activity from Bacillus Stearothermophilus DNA polymerase in the presence of magnesium

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13543799P 1999-05-22 1999-05-22
US60/135,437 1999-05-22

Publications (2)

Publication Number Publication Date
WO2000071739A1 WO2000071739A1 (fr) 2000-11-30
WO2000071739A9 true WO2000071739A9 (fr) 2002-07-04

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Application Number Title Priority Date Filing Date
PCT/US2000/013960 Ceased WO2000071739A1 (fr) 1999-05-22 2000-05-19 Activite de transcription inverse de l'adn polymerase issu de bacillus stearothermophilus, en presence de magnesium

Country Status (5)

Country Link
EP (1) EP1185680A4 (fr)
JP (1) JP2003500063A (fr)
AU (1) AU765313B2 (fr)
CA (1) CA2374494A1 (fr)
WO (1) WO2000071739A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040009486A1 (en) 1999-10-29 2004-01-15 Sorge Joseph A. Compositions and methods utilizing DNA polymerases
US6436677B1 (en) * 2000-03-02 2002-08-20 Promega Corporation Method of reverse transcription
US7179590B2 (en) * 2000-04-18 2007-02-20 Roche Molecular Systems, Inc High temperature reverse transcription using mutant DNA polymerases
US9689042B2 (en) 2012-09-07 2017-06-27 The Regents Of The University Of California Use of glycolytic pathways for inhibiting or measuring oncogenic signaling
CN105176971A (zh) * 2015-08-14 2015-12-23 青岛科技大学 Bst DNA聚合酶在RNA扩增中的应用
CN106399299B (zh) * 2016-09-29 2019-01-29 华南理工大学 一种通过点突变提高大片段嗜热脂肪地芽孢杆菌dna聚合酶活性的方法及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100078A (en) * 1994-04-01 2000-08-08 Gen-Probe Incorporated Purified DNA polymerase from bacillus stearothermophilus ATCC 12980
US5830714A (en) * 1996-04-17 1998-11-03 Molecular Biology Resources, Inc. Biologically active fragment of bacillus stearothermophilus DNA polymerase

Also Published As

Publication number Publication date
AU5036100A (en) 2000-12-12
CA2374494A1 (fr) 2000-11-30
EP1185680A1 (fr) 2002-03-13
JP2003500063A (ja) 2003-01-07
AU765313B2 (en) 2003-09-18
WO2000071739A1 (fr) 2000-11-30
EP1185680A4 (fr) 2004-08-18

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