US20020004230A1 - Stabilized aqueous nucleoside triphosphate solution - Google Patents

Stabilized aqueous nucleoside triphosphate solution Download PDF

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Publication number
US20020004230A1
US20020004230A1 US09/308,034 US30803499A US2002004230A1 US 20020004230 A1 US20020004230 A1 US 20020004230A1 US 30803499 A US30803499 A US 30803499A US 2002004230 A1 US2002004230 A1 US 2002004230A1
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aqueous solution
stable aqueous
value
solution
mmol
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US09/308,034
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Hans-Georg Ihlenfeldt
Axel Schmidt
Klaus Muhlegger
Volker Leitenberger
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Roche Diagnostics GmbH
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Assigned to ROCHE DIAGNOSTICS GMBH reassignment ROCHE DIAGNOSTICS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MUHLEGGER, KLAUS, LEITENBERGER, VOLKER, IHLENFELDT, HANS-GEORG, SCHMIDT, AXEL
Priority to US10/025,826 priority Critical patent/US6916616B2/en
Publication of US20020004230A1 publication Critical patent/US20020004230A1/en
Abandoned legal-status Critical Current

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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
    • 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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6846—Common amplification features
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention concerns stable aqueous solutions containing nucleoside triphosphates in which the solution has a pH value above 7.5.
  • Nucleoside triphosphates such as ribonucleoside, deoxynucleoside and dideoxynucleoside triphosphates have a variety of uses in the field of biochemistry and molecular biology. Most of the applications relate to reactions which synthesize or replicate DNA and RNA such as the reverse transcriptase polymerase chain reaction (RT-PCR), cycle sequencing and nick translation. In the case of RT-PCR, DNA chains are synthesized in the 5′-3′ direction by for example reverse transcriptase whereby an RNA strand serves as the template. Certain NTPs such as dideoxynucleoside triphosphates (dd-NTP) can be used as chain terminators in the sequencing of DNA.
  • dd-NTP dideoxynucleoside triphosphates
  • d-NTP deoxynucleoside triphosphates
  • PCR polymerase chain reaction
  • NTP solutions A disadvantage of the current, i.e. commercially available, NTP solutions is in particular the instability of the NTPs during storage or thermal stress.
  • the NTPs have a tendency to decompose over time to form the corresponding diphosphates and monophosphates.
  • the triphosphate content decreases especially at higher temperatures.
  • the triphosphate content already decreases by ca. 2-3% within ten days at a pH value of ca. 7.5 and a temperature of 35° C. In contrast at room temperature the triphosphate content is observed to decrease by only ca. 1% after six weeks.
  • the decomposition of the triphosphates in aqueous solution limits the shelf life of the NTP solutions.
  • the object of the present invention was to provide a stabilized aqueous solution containing NTPs without the addition of any stabilizers.
  • nucleotide triphosphates include ribonucleoside triphosphates, deoxynucleotide and dideoxynucleotide triphosphates wherein the five naturally occurring as well as modified bases such as isoguanine, deaza compounds and derivatives thereof come into consideration as bases.
  • nucleoside triphosphates can be labelled with reporter groups.
  • the solutions according to the invention have a pH value in a range of more than 7.5 to a maximum of 11.
  • a pH value of ca. 8 to 10 proved to be particularly advantageous.
  • the pH value can be set by adding a base (e.g. NaOH, KOH, LiOH) as well as by adding a buffer (e.g Tris buffer, Na carbonate buffer, phosphate buffer).
  • the concentration of the NTP solution is preferably between ca. 2 mmol/l and 200 mmol/l.
  • a concentration of the NTPs of ca. 100 to 150 mmol/l is particularly preferred.
  • Stable d-NTP solutions are a particularly important feature of this invention.
  • the stability of these solutions appears to be advantageous especially with regard to an application in the polymerase chain reaction.
  • the pH value of the d-NTP solution is above ca. 7.5 and below ca. pH 11.
  • a pH value between ca. 8 and 10 proved to be particularly advantageous.
  • the concentration of the stable d-NTP solution is between 2 mmol/l and 200 mmol/l.
  • a concentration of the d-NTPs of 100 to 150 mmol/l is particularly preferred.
  • the stable NTP solutions according to the invention can be used for all DNA and RNA synthesizing and DNA and RNA replicating reactions.
  • the stable NTP solution according to the invention can also be used for RT-PCR, for nick translation, random priming and for sequencing (cycle sequencing).
  • the stable NTP solutions according to the invention proved to be advantageous with regard to a longer duration of use of the NTPs. That means that the stable solutions according to the invention can be stored for a considerably longer period than the previously used d-NTP solutions.
  • FIG. 1 Decrease of the d-GTP content The decrease of the d-GTP concentration at a temperature of 35° C. was monitored over a period of 140 days at pH values of 7.5, 7.9 and 8.4.
  • FIG. 2 Decrease of the d-CTP content The decrease of the d-CTP concentration at a temperature of 35° C. was monitored over a period of 90 days at pH values of 7.5, 7.9 and 8.3.
  • FIG. 3 Decrease of the d-TTP content The decrease of the d-TTP concentration at a temperature of 35° C. was monitored over a period of 90 days at pH values of 7.5, 7.9 and 8.3.
  • FIG. 4 Decrease of the d-UTP content The decrease of the d-UTP concentration at a temperature of 35° C. was monitored over a period of 90 days at pH values of 7.5, 7.9 and 8.3.
  • FIG. 5 Decrease of the d-ATP content The decrease of the d-ATP concentration at a temperature of 35° C. was monitored over a period of 65 days at pH values of 7.5, 7.9 and 8.4.
  • FIG. 6 Triphosphate content in relation to the pH value
  • FIG. 7 pH dependence of the stability of UTP
  • FIG. 8 pH dependence of the stability of UDP
  • FIG. 9 pH dependence of the stability of ATP
  • FIG. 10 pH dependence of the stability of ADP
  • FIG. 11 pH dependence of the stability of 7-deazadeoxy-GTP
  • FIG. 12 pH dependence of the stability of 7-deazadeoxy-GTP
  • d-NTPs were purified by anion chromatography with the aid of a salt gradient and desalted by reverse osmosis. This is followed by an ultrafiltration (exclusion limit 1000-5000 D) to remove DNAses/RNAses. The concentration of the solution is then adjusted with sterile water to typically 100 mM. The pH value is adjusted to the corresponding pH value (>7.5) by the addition of bases (alkali/alkaline earth/ammonium hydroxide; amines) usually NaOH.
  • bases alkali/alkaline earth/ammonium hydroxide; amines
  • d-NTP solutions at a concentration of 100 to 110 mmol/l were adjusted to pH values between 7.5 and 8.3 with sodium hydroxide solution.
  • the sample was stored at 35° C., 22° C., 4° C. and ⁇ 20° C. Aliquots were removed at various time points and the purity was examined by means of HPLC. The relative amount of the tri, di and monophosphate as well as of the free base was determined by integrating the areas.
  • d-NTP solutions (dCTP, dTTP, dUTP) at a concentration of 100 to 110 mmol/l were adjusted to pH values between 7.5 and 12 (d-ATP, dGTP not pH 7.9 and 8.3) with sodium hydroxide solution.
  • the sample was stressed for 35 days at 35° C. and subsequently the purity was examined by means of HPLC. The relative amount of the tri, di and monophosphate as well as of the free base was determined by integrating the areas.
  • the stabilization was estimated by the following formula from three independent stress experiments of d-NTPs at pH 8.3 and 7.5 at 35° C. in which samples were taken at intervals between 7 and 89 days.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US09/308,034 1996-11-14 1997-11-11 Stabilized aqueous nucleoside triphosphate solution Abandoned US20020004230A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/025,826 US6916616B2 (en) 1996-11-14 2001-12-19 Stabilized aqueous nucleoside triphosphate solutions

Applications Claiming Priority (2)

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DE19647055.2 1996-11-14
DE19647055A DE19647055A1 (de) 1996-11-14 1996-11-14 Stabilisierte wässrige Nukleosidtriphosphat-Lösungen

Related Parent Applications (1)

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US (2) US20020004230A1 (de)
EP (1) EP0941370B1 (de)
JP (1) JP2001503764A (de)
KR (1) KR100489496B1 (de)
CN (1) CN1238012A (de)
AR (1) AR011276A1 (de)
AT (1) ATE219152T1 (de)
AU (1) AU737546B2 (de)
CA (1) CA2277198C (de)
CZ (1) CZ167899A3 (de)
DE (2) DE19647055A1 (de)
IL (1) IL129684A0 (de)
NZ (1) NZ335426A (de)
PL (1) PL333362A1 (de)
WO (1) WO1998021362A2 (de)
ZA (1) ZA9710230B (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170191044A1 (en) * 2011-04-11 2017-07-06 Roche Molecular Systems, Inc. Dna polymerases with improved activity

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DE19647055A1 (de) 1996-11-14 1998-05-20 Boehringer Mannheim Gmbh Stabilisierte wässrige Nukleosidtriphosphat-Lösungen
EP1706512B1 (de) 2004-01-23 2008-12-17 BioMerieux, Inc. Neuartiges nukleotidgemisch für ein verbessertes nukleinsäureamplifikationsverfahren
RU2259401C1 (ru) * 2004-04-27 2005-08-27 Общество с ограниченной ответственностью "Компания "Биоком" (ООО "Компания "Биоком") Сухая смесь реагентов для полимеразной цепной реакции и способ проведения пцр-анализа
US20070114476A1 (en) * 2005-11-04 2007-05-24 Williams Christopher P Low radiocarbon nucleotide and amino acid dietary supplements
EP2158462A4 (de) 2007-05-03 2010-05-05 Radiocarb Genetics Inc Nahrungsergänzungen mit wenig radiokohlenstoff und herstellungsverfahren dafür
US20100222294A1 (en) * 2009-02-27 2010-09-02 Duska Scientific Co. Formulations of ATP and Analogs of ATP
DE102010038330A1 (de) 2010-07-23 2012-03-01 Aj Innuscreen Gmbh Verfahren, Vorrichtung und Testkit für Molekularbiologische Reaktionen

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US4965188A (en) * 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US5374553A (en) * 1986-08-22 1994-12-20 Hoffmann-La Roche Inc. DNA encoding a thermostable nucleic acid polymerase enzyme from thermotoga maritima
FR2674253B1 (fr) * 1991-03-19 1995-10-20 Diagnostics Pasteur Composition lyophilisee pour la multiplication de sequences d'acides nucleiques.
US5686058A (en) * 1992-04-30 1997-11-11 Amersham International Plc Radiolabelled nucleotide formulations stored in an unfrozen state
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DE4336266A1 (de) * 1993-10-23 1995-04-27 Boehringer Mannheim Gmbh Stabilisierte flüssige Mischungen für die Markierung von Nukleinsäuren
US5643723A (en) * 1994-05-26 1997-07-01 Roche Molecular Systems, Inc. Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170191044A1 (en) * 2011-04-11 2017-07-06 Roche Molecular Systems, Inc. Dna polymerases with improved activity
US10597644B2 (en) * 2011-04-11 2020-03-24 Roche Molecular Systems, Inc. DNA polymerases with improved activity

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NZ335426A (en) 1999-10-28
EP0941370B1 (de) 2002-06-12
US20020119534A1 (en) 2002-08-29
US6916616B2 (en) 2005-07-12
WO1998021362A3 (de) 1998-07-02
JP2001503764A (ja) 2001-03-21
CA2277198A1 (en) 1998-05-22
KR100489496B1 (ko) 2005-05-16
DE59707529D1 (de) 2002-07-18
WO1998021362A2 (de) 1998-05-22
CN1238012A (zh) 1999-12-08
ATE219152T1 (de) 2002-06-15
AR011276A1 (es) 2000-08-16
EP0941370A2 (de) 1999-09-15
PL333362A1 (en) 1999-12-06
CZ167899A3 (cs) 1999-10-13
ZA9710230B (en) 1999-05-11
CA2277198C (en) 2008-10-07
AU5321398A (en) 1998-06-03
DE19647055A1 (de) 1998-05-20
AU737546B2 (en) 2001-08-23
IL129684A0 (en) 2000-02-29
KR20000053066A (ko) 2000-08-25

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