CA2445864A1 - A hot start amplification process using pyrophosphatase enzyme - Google Patents
A hot start amplification process using pyrophosphatase enzyme Download PDFInfo
- Publication number
- CA2445864A1 CA2445864A1 CA002445864A CA2445864A CA2445864A1 CA 2445864 A1 CA2445864 A1 CA 2445864A1 CA 002445864 A CA002445864 A CA 002445864A CA 2445864 A CA2445864 A CA 2445864A CA 2445864 A1 CA2445864 A1 CA 2445864A1
- Authority
- CA
- Canada
- Prior art keywords
- reaction mixture
- ppase
- pyrophosphate
- pyrophosphatase
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract 23
- 230000003321 amplification Effects 0.000 title claims abstract 17
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract 17
- 108010009413 Pyrophosphatases Proteins 0.000 title claims abstract 10
- 102000009609 Pyrophosphatases Human genes 0.000 title claims abstract 10
- 239000011541 reaction mixture Substances 0.000 claims abstract 14
- 238000006243 chemical reaction Methods 0.000 claims abstract 13
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical class [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims abstract 11
- 102000039446 nucleic acids Human genes 0.000 claims abstract 3
- 108020004707 nucleic acids Proteins 0.000 claims abstract 3
- 150000007523 nucleic acids Chemical class 0.000 claims abstract 3
- 235000011180 diphosphates Nutrition 0.000 claims 5
- 238000003752 polymerase chain reaction Methods 0.000 claims 5
- 239000003153 chemical reaction reagent Substances 0.000 claims 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims 2
- 239000012634 fragment Substances 0.000 claims 2
- 102000040430 polynucleotide Human genes 0.000 claims 2
- 108091033319 polynucleotide Proteins 0.000 claims 2
- 239000002157 polynucleotide Substances 0.000 claims 2
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical group [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 claims 2
- 241000567139 Aeropyrum pernix Species 0.000 claims 1
- 101900352645 Aeropyrum pernix Inorganic pyrophosphatase Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 108010009595 Inorganic Pyrophosphatase Proteins 0.000 claims 1
- 102000009617 Inorganic Pyrophosphatase Human genes 0.000 claims 1
- 241000205156 Pyrococcus furiosus Species 0.000 claims 1
- 108010006785 Taq Polymerase Proteins 0.000 claims 1
- 241000205188 Thermococcus Species 0.000 claims 1
- 101900314195 Thermococcus litoralis Inorganic pyrophosphatase Proteins 0.000 claims 1
- 241000589596 Thermus Species 0.000 claims 1
- 241000557720 Thermus brockianus Species 0.000 claims 1
- 241000589499 Thermus thermophilus Species 0.000 claims 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 230000029087 digestion Effects 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 238000009830 intercalation Methods 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 239000000523 sample Substances 0.000 claims 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 claims 1
Classifications
-
- 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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- 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
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/50—Other enzymatic activities
- C12Q2521/525—Phosphatase
-
- 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
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/186—Modifications characterised by incorporating a non-extendable or blocking moiety
-
- 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
- C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
- C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
- C12Q2549/101—Hot start
Abstract
A method for conducting a nucleic acid amplification reaction, said method comprising forming an amplification reaction mixture in the presence of sufficient of a pyrophosphate salt to prevent primer extension taking place, digesting said pyrophosphate salt with a pyrophosphatase enzyme (PPase), and subjecting said reaction mixture to conditions such that an amplification reaction may proceed. This can be used as a "hot start" amplification.
Particular novel pyrophosphatase enzymes for use in the method are also described and claimed.
Particular novel pyrophosphatase enzymes for use in the method are also described and claimed.
Claims (26)
1. A method for conducting a nucleic acid amplification reaction, said method comprising forming an amplification reaction mixture in the presence of sufficient of a pyrophosphate salt to prevent primer extension taking place, digesting said pyrophosphate salt with a pyrophosphatase enzyme (PPase), and subjecting said reaction mixture to conditions such that an amplification reaction may proceed.
2. A method according to claim 1 wherein the amplification reaction is a polymerase chain reaction (PCR) and the reaction mixture contains reagents suitable for conducting such a reaction.
3. A method according to claim 1 or claim 2 wherein the reaction mixture contains a DNA polymerase which is selected from Thermus aquaticus polymerase (Taq), Thermus thermophilus polymerase (Tth), Thermus species NH polymerase (TspNH), Thermus brockianus polymerase (Tbr), Pyrococcus furiosus polymerase (Pfu), 9°N7 exo-DNa polymerase, and Thermococcus literalis DNA polymerase.
4. A method according to any one of the preceding claims wherein the inorganic pyrophosphate is an alkali earth metal pyrophpsphate.
5. A method according to claim 4 wherein the inorganic pyrophosphate is tetrasodium pyrophosphate of formula Na4P2O7.
6. A method according to any one of the preceding claims wherein the pyrophosphate is present in the reaction mixture at a concentration of at least 0.5mM.
7. A method according to claim 6 wherein the pyrophosphate is present at a concentration of from 1-10mM.
8. A method according to any one of the preceding claims wherein the said digestion is effected using a thermostable PPase.
9. A method according to claim 8 wherein the thermostable PPase is Sulfolbus acidicaldarius inorganic pyrophosphatase, (Sac PPase), thermococcus litoralis inorganic pyrophosphatase or Aeropyrum pernix inorganic pyrophosphatase.
10. A method according to claim 8 or claim 9 wherein the thermostable PPase is added to the reaction mixture on formation thereof.
11. A method according to claim 10 which includes a incubation step prior to the amplification reaction at elevated temperature in order to allow the PPase to digest inorganic pyrophosphate present.
12. A method according to any one of the preceding claims wherein the PPase is added to the reaction mixture at a concentration of at least 0.04u per 50µL PCR reaction mixture.
13. A method according to claim 12 wherein the PPase is added to the reaction mixture at a concentration of 0.08u per 50µL
PCR reaction mixture.
PCR reaction mixture.
14. A method according to claim 12 or claim 13 wherein the PPase is added to the reaction mixture at a concentration of from 0.2 -10u per 50µL PCR reaction mixture.
15. A kit for conducting an amplification reaction, said kit comprising a pyrophosphate salt, an pyrophosphatase enzyme, and optionally one or more reagents required for use in an amplification reaction.
16. A kit according to claim 15 which further comprises one or more primers necessary to carry out amplification of a particular target nucleic acid.
17. A kit according to claim 15 or claim 16 which further includes one or more fluorescently labelled reagents.
18. A kit according to claim 17 wherein the fluorescently labelled reagents are selected from one or more of an intercalating dye, a fluorescently labelled probe, a fluorescently labelled primer or a fluorescently labelled nucleotide.
19. The use of a pyrophosphate salt in a method for carrying out amplification reactions as claimed in any one of claims 1 to 14.
20. The use of a pyrophosphatase enzyme in a method for carrying out amplification reactions as claimed in any one of claims 1 to 14.
22. A pyrophosphatase enzyme isolated from Aeropyrum pernix.
22. A pyrophosphatase enzyme encoded by the polynucleotide sequence as shown in SEQ ID NO.26 or a variant or fragment thereof.
23. A pyrophosphatase enzyme comprising the amino acid sequence as shown in SEQ ID NO. 25 or a variant or fragment thereof.
24. An isolated polynucleotide which encodes an enzyme according to claim 22 or claim 23.
25. The use of a pyrophosphatase enzyme as claimed in any one of claims 21 to 23 in a method for carrying out amplification reactions as claimed in any one of claims 1 to 14.
26. A method for conducting an amplification reaction substantially as herein before described with reference to the Examples.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB0110501.4A GB0110501D0 (en) | 2001-04-30 | 2001-04-30 | Amplification process |
| GB0110501.4 | 2001-04-30 | ||
| PCT/GB2002/001861 WO2002088387A2 (en) | 2001-04-30 | 2002-04-22 | Amplification process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2445864A1 true CA2445864A1 (en) | 2002-11-07 |
| CA2445864C CA2445864C (en) | 2011-08-02 |
Family
ID=9913697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2445864A Expired - Fee Related CA2445864C (en) | 2001-04-30 | 2002-04-22 | A hot start amplification process using pyrophosphatase enzyme |
Country Status (14)
| Country | Link |
|---|---|
| US (2) | US6951744B2 (en) |
| EP (1) | EP1390532B1 (en) |
| JP (1) | JP4177118B2 (en) |
| KR (2) | KR101039563B1 (en) |
| CN (1) | CN1318604C (en) |
| AR (1) | AR035238A1 (en) |
| AT (1) | ATE502118T1 (en) |
| AU (1) | AU2002249444B2 (en) |
| CA (1) | CA2445864C (en) |
| DE (1) | DE60239456D1 (en) |
| GB (2) | GB0110501D0 (en) |
| NZ (1) | NZ528919A (en) |
| TW (1) | TWI323284B (en) |
| WO (1) | WO2002088387A2 (en) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0112868D0 (en) | 2001-05-25 | 2001-07-18 | Secr Defence | Detection system |
| CN1500887A (en) * | 2002-10-01 | 2004-06-02 | 松下电器产业株式会社 | Method for detecting primer elongation reaction, method and apparatus for distinguishing kinds of basic groups |
| EP1634965B1 (en) | 2004-09-09 | 2010-01-20 | Roche Diagnostics GmbH | Real time PCR with the addition of pyrophosphatase |
| US20060051796A1 (en) * | 2004-09-09 | 2006-03-09 | Inga Boell | Real time PCR with the addition of pyrophosphatase |
| GB0502010D0 (en) * | 2005-02-01 | 2005-03-09 | Enigma Diagnostics Ltd | Biochemical reagents and their uses |
| DE602007013223D1 (en) | 2006-06-01 | 2011-04-28 | Trilink Biotechnologies San Diego | CHEMICALLY MODIFIED OLIGONUCLEOTIDE PRIMERS FOR NUCLEIC ACID AMPLIFICATION |
| KR101098764B1 (en) | 2007-10-29 | 2011-12-26 | (주)바이오니아 | Dried Composition for hot-start PCR with Long-Term Stability |
| US8133669B2 (en) | 2008-05-27 | 2012-03-13 | Trilink Biotechnologies | Chemically modified nucleoside 5′-triphosphates for thermally initiated amplification of nucleic acid |
| EP2566959A1 (en) * | 2010-04-30 | 2013-03-13 | Roche Diagniostics GmbH | System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus |
| US20110312763A1 (en) * | 2010-06-17 | 2011-12-22 | Geneasys Pty Ltd | Genetic analysis loc with in-loc storage of all required reagents |
| KR101870311B1 (en) * | 2012-03-09 | 2018-06-25 | (주)바이오니아 | Compositions for hot start reverse transcription reaction or hot start reverse transcription polymerase chain reaction |
| EP2861757B1 (en) | 2012-06-14 | 2019-11-06 | Life Technologies Corporation | Novel compositions, methods and kits for polymerase chain reaction (pcr) |
| GB201301457D0 (en) | 2013-01-28 | 2013-03-13 | Fluorogenics Ltd | Freeze-dried composition |
| JP6467829B2 (en) * | 2014-09-03 | 2019-02-13 | 東洋紡株式会社 | Improved RT-PCR reaction |
| CN104862288B (en) * | 2015-05-08 | 2019-10-29 | 苏州大学 | A kind of synthesis of New temperature responsiveness inorganic pyrophosphate enzyme conjugates and its application in enhancing polymerase chain reaction |
| US10626383B2 (en) | 2016-01-15 | 2020-04-21 | Thermo Fisher Scientific Baltics Uab | Thermophilic DNA polymerase mutants |
| WO2019002178A1 (en) | 2017-06-26 | 2019-01-03 | Thermo Fisher Scientific Baltics Uab | Thermophilic dna polymerase mutants |
| JP7234598B2 (en) * | 2018-01-26 | 2023-03-08 | 東ソー株式会社 | Nucleic acid amplification reagent and nucleic acid amplification control method using the reagent |
| CN113481180A (en) * | 2021-07-05 | 2021-10-08 | 吉林大学 | Alkaline thermophilic inorganic pyrophosphatase and application thereof in enhancing polymerase chain reaction and UDP-galactose synthesis reaction |
Family Cites Families (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4868103A (en) | 1986-02-19 | 1989-09-19 | Enzo Biochem, Inc. | Analyte detection by means of energy transfer |
| CH670709A5 (en) * | 1986-04-24 | 1989-06-30 | Univ Moskovsk | |
| US5498523A (en) * | 1988-07-12 | 1996-03-12 | President And Fellows Of Harvard College | DNA sequencing with pyrophosphatase |
| HUT61054A (en) * | 1989-04-12 | 1992-11-30 | Harvard College | Improved process and test kit for primary extension reactions |
| KR100236506B1 (en) | 1990-11-29 | 2000-01-15 | 퍼킨-엘머시터스인스트루먼츠 | Apparatus for polymerase chain reaction |
| FI923911A (en) * | 1992-09-01 | 1994-03-02 | Vsevolod Kiselev | DNA molecules in vitro syntheses |
| US5565339A (en) | 1992-10-08 | 1996-10-15 | Hoffmann-La Roche Inc. | Compositions and methods for inhibiting dimerization of primers during storage of polymerase chain reaction reagents |
| US5491063A (en) | 1994-09-01 | 1996-02-13 | Hoffmann-La Roche Inc. | Methods for in-solution quenching of fluorescently labeled oligonucleotide probes |
| US5773258A (en) | 1995-08-25 | 1998-06-30 | Roche Molecular Systems, Inc. | Nucleic acid amplification using a reversibly inactivated thermostable enzyme |
| US5665551A (en) * | 1995-09-13 | 1997-09-09 | Roche Molecular Systems, Inc. | Purified nucleic acid encoding a thermostable pyrophosphatase |
| US6291164B1 (en) * | 1996-11-22 | 2001-09-18 | Invitrogen Corporation | Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate |
| GB9716052D0 (en) | 1996-12-06 | 1997-10-01 | Secr Defence | Reaction vessels |
| GB9725197D0 (en) | 1997-11-29 | 1998-01-28 | Secr Defence | Detection system |
| GB9725237D0 (en) | 1997-11-29 | 1998-01-28 | Secr Defence | Amplification system |
| GB9803382D0 (en) | 1998-02-19 | 1998-04-15 | Secr Defence | Detection system |
| US6183998B1 (en) | 1998-05-29 | 2001-02-06 | Qiagen Gmbh Max-Volmer-Strasse 4 | Method for reversible modification of thermostable enzymes |
| GB9812768D0 (en) | 1998-06-13 | 1998-08-12 | Zeneca Ltd | Methods |
| KR100292883B1 (en) * | 1999-02-09 | 2001-06-15 | 박한오 | Hot Start PCR Mixture Comprising Pyrophosphate and Pyrophosphatase |
| JP4989004B2 (en) | 2000-02-23 | 2012-08-01 | シティ・オブ・ホープ | Pyrophosphate degradation-activated polymerization (PAP): adaptation to allyl-specific amplification and nucleic acid sequencing |
| KR20050005626A (en) * | 2003-07-07 | 2005-01-14 | 주식회사 포스코 | improvement method of charging coal bulk density in a coke oven |
-
2001
- 2001-04-30 GB GBGB0110501.4A patent/GB0110501D0/en not_active Ceased
-
2002
- 2002-03-28 TW TW091106126A patent/TWI323284B/en not_active IP Right Cessation
- 2002-04-22 NZ NZ528919A patent/NZ528919A/en unknown
- 2002-04-22 CA CA2445864A patent/CA2445864C/en not_active Expired - Fee Related
- 2002-04-22 WO PCT/GB2002/001861 patent/WO2002088387A2/en active Application Filing
- 2002-04-22 EP EP02718374A patent/EP1390532B1/en not_active Expired - Lifetime
- 2002-04-22 KR KR1020037014191A patent/KR101039563B1/en not_active IP Right Cessation
- 2002-04-22 CN CNB028132726A patent/CN1318604C/en not_active Expired - Fee Related
- 2002-04-22 AU AU2002249444A patent/AU2002249444B2/en not_active Ceased
- 2002-04-22 KR KR1020097025792A patent/KR20100005245A/en not_active Application Discontinuation
- 2002-04-22 AT AT02718374T patent/ATE502118T1/en not_active IP Right Cessation
- 2002-04-22 DE DE60239456T patent/DE60239456D1/en not_active Expired - Lifetime
- 2002-04-22 JP JP2002585667A patent/JP4177118B2/en not_active Expired - Fee Related
- 2002-04-22 GB GB0209052A patent/GB2377991B/en not_active Expired - Fee Related
- 2002-04-29 AR ARP020101570A patent/AR035238A1/en unknown
- 2002-04-30 US US10/135,807 patent/US6951744B2/en not_active Expired - Fee Related
-
2005
- 2005-08-17 US US11/205,667 patent/US7449312B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US6951744B2 (en) | 2005-10-04 |
| KR20100005245A (en) | 2010-01-14 |
| WO2002088387A2 (en) | 2002-11-07 |
| ATE502118T1 (en) | 2011-04-15 |
| GB2377991B (en) | 2004-01-28 |
| CN1318604C (en) | 2007-05-30 |
| DE60239456D1 (en) | 2011-04-28 |
| KR101039563B1 (en) | 2011-06-09 |
| AR035238A1 (en) | 2004-05-05 |
| US7449312B2 (en) | 2008-11-11 |
| JP2004524853A (en) | 2004-08-19 |
| NZ528919A (en) | 2005-07-29 |
| WO2002088387A3 (en) | 2003-12-11 |
| US20030049655A1 (en) | 2003-03-13 |
| GB0209052D0 (en) | 2002-05-29 |
| EP1390532B1 (en) | 2011-03-16 |
| EP1390532A2 (en) | 2004-02-25 |
| KR20040015216A (en) | 2004-02-18 |
| CN1522305A (en) | 2004-08-18 |
| US20060057617A1 (en) | 2006-03-16 |
| JP4177118B2 (en) | 2008-11-05 |
| AU2002249444B2 (en) | 2007-07-12 |
| CA2445864C (en) | 2011-08-02 |
| GB0110501D0 (en) | 2001-06-20 |
| TWI323284B (en) | 2010-04-11 |
| GB2377991A (en) | 2003-01-29 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20180423 |