WO2001092569A2 - Formulation for polymerase chain reaction and vessel containing same - Google Patents
Formulation for polymerase chain reaction and vessel containing same Download PDFInfo
- Publication number
- WO2001092569A2 WO2001092569A2 PCT/GB2001/002265 GB0102265W WO0192569A2 WO 2001092569 A2 WO2001092569 A2 WO 2001092569A2 GB 0102265 W GB0102265 W GB 0102265W WO 0192569 A2 WO0192569 A2 WO 0192569A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- formulation
- vessel
- polymerase
- chain reaction
- effecting
- Prior art date
Links
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
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the present invention relates to formulations for use in effecting a Polymerase Chain Reaction and also to vessels containing such a formulation and which are intended for use in conducting such a reaction.
- PCR Polymerase Chain Reaction
- This lyophilised mixture has the advantage that it simplifies the multi-step PCR manipulation in that all components (except target) for effecting amplification are included in the pre-prepared mixture such that all that is required is addition of an aqueous sample containing (or potentially' containing) the target. Furthermore, the water soluble dye facilitates identification of complete mixing of the PCR reagent and test sample and saves the trouble of adding a sample loading buffer which is otherwise required for analysis of PCR products. As a result, the formulations of US-A-5 861 251 provide the advantage of avoiding carry-over contamination into the PCR reaction mix. However detection of the amplified product is effected by running the product mixture on a gel. This necessities opening of the tube, to apply the product mixture to the gel, thus once again giving rise to the possibility of cross-contamination.
- a formulation for use in effecting a Polymerase Chain Reaction comprising a dried composition of reagents including reaction buffer, dNTPs, at least two primers and a polymerase and said formulation being re-hydratable to be capable of effecting amplification of a target nucleic acid sequence of interest characterised in that the formulation incorporates a fluorescent reporter molecule capable of reporting by homologous detection the presence of amplified nucleic acid produced by the Polymerase Chain Reaction..
- the formulation of the invention is such that only a single addition of aqueous target sample to the formulation is required to produce an aqueous reaction mixture containing all necessary components for PCR amplification of target nucleic acid sequence.
- the formulation of the invention does however have the significant additional advantage that the presence, in the formulation, of the fluorescent reporter molecule means homologous detection may be used.
- the progress of the reaction may be followed by real-time detection techniques avoiding the need for post-reaction manipulation of the product mixture (e.g. transferring the mixture to a gel, or even opening a vessel in which the product mixture is contained) thereby avoiding any possibility of cross-contamination.
- This has dramatic consequences for the set-up of laboratories that PERform PCR-based diagnostic reactions as, currently, extreme care has to be taken during the performance of the reaction to prevent cross-contamination.
- no particular contamination controls would be needed other than those routine in a molecular biology laboratory.
- There are also additional benefits including having much more defined reaction conditions (as essentially all the reactants could come from the same batch, convenience, longer shelf life etc).
- the invention also provides, according to a second aspect thereof, a vessel (e.g. a reaction tube) containing a pre-measured amount of the formulation of the invention.
- a vessel e.g. a reaction tube
- the vessels may be provided with a suitable closure element and supplied to end users who, after removal of the closure element merely, need only to add the aqueous sample and then re-close the vessel.
- the end user may be a person in a laboratory where the PCR reaction is then effected. Alternatively the end user may be out "on-site" collecting samples which can then be added to the vessel as soon as collected, the vessel then being sent to a laboratory for conducting the PCR reaction.
- the inner surface of the vessel (adjacent the mouth thereof) and the outer surface of the closure element may be provided with inter-engageable formations allowing insertion of the closure element into the vessel but preventing withdrawal therefrom.
- inter-engagable formations should be positioned such that the closure element is capable of being removable provided that it has not been inserted into the vessel beyond a certain degree.
- the dried composition may be incorporated into the vessel and the closure element removably applied thereto. Subsequently the closure element may be removed to permit addition of the sample and then subsequently inserted sufficiently far into the vessel so that it becomes non-removable.
- the formulation of the invention may be prepared by lyophilisation of an aqueous solution of the required components, e.g. by lyophilisation using the procedures disclosed in US-A-5 861 251.
- the solution includes a stabiliser which may for example be glucose, glucitol or trehalose.
- the dried formulation of the invention may be such that, per ml of reconstituted reaction medium, it comprises: Component Amount
- Stabiliser e.g. trehalose 0.1-15% w/w
- the PCR reaction may be conducted by procedures well known in the art, e.g. using thermal cycling.
- the fluorescent reporter molecule included in the formulation of the invention may for example be one which reports a change in the amount of double stranded DNA present in the reaction, e.g. an intercalating dye such as Ethidium Bromide, CyBr- Green or PicoGreen.
- the fluorescent reporter molecule may be one which works in conjunction with a quencher moiety so as to be capable of reporting on the presence of specific nucleotide sequences in the mixture and may, for example, be a TaqMan probe, Molecular Beacon, Sunrise primer and Scorpion primer (Registered Trade Marks).
- the polymerase may be a DNA polymerase and may be a thermally stable polymerase, e.g. Taq polymerase.
- Taq polymerase e.g. a thermally stable polymerase
- the polymerase in the formulation of the invention is a "hot-start" polymerase.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01936610A EP1290223A2 (en) | 2000-05-31 | 2001-05-23 | Formulation for polymerase chain reaction and vessel containing same |
AU2001262483A AU2001262483A1 (en) | 2000-05-31 | 2001-05-23 | Formulation for polymerase chain reaction and vessel containing same |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0013043A GB0013043D0 (en) | 2000-05-31 | 2000-05-31 | Formulation for polymerase chain reaction and vessel containing same |
GB0013043.5 | 2000-05-31 | ||
GB0013863A GB0013863D0 (en) | 2000-06-07 | 2000-06-07 | Formulation for polymerase chain reaction and vessel containing same |
GB0013863.6 | 2000-06-07 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001092569A2 true WO2001092569A2 (en) | 2001-12-06 |
WO2001092569A3 WO2001092569A3 (en) | 2002-03-28 |
Family
ID=26244374
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2001/002265 WO2001092569A2 (en) | 2000-05-31 | 2001-05-23 | Formulation for polymerase chain reaction and vessel containing same |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1290223A2 (en) |
AU (1) | AU2001262483A1 (en) |
WO (1) | WO2001092569A2 (en) |
Cited By (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1350855A2 (en) * | 2002-04-01 | 2003-10-08 | Agilent Technologies, Inc. - a Delaware corporation - | Array based hybridization assays |
EP1498492A1 (en) * | 2003-07-15 | 2005-01-19 | Lukas Bestmann | Apparatus for the preparation of samples |
WO2005118849A1 (en) * | 2004-06-04 | 2005-12-15 | Abacus Diagnostica Oy | Method for stabilizing assay reagents, reagent container with stabilized assay reagents and use thereof |
WO2006000648A1 (en) * | 2004-06-29 | 2006-01-05 | Wallac Oy | Integrated nucleic acid analysis |
US7776530B2 (en) | 2004-06-29 | 2010-08-17 | Wallac Oy | Integrated nucleic acid analysis |
EP2210955A3 (en) * | 2006-05-23 | 2010-09-15 | Molecular Detection, Inc. | Ambient temperature stable kits for molecular diagnostics |
WO2012010708A1 (en) * | 2010-07-23 | 2012-01-26 | Aj Innuscreen Gmbh | Method, device and test kit for molecular-biological reactions |
EP2574931A1 (en) | 2011-09-29 | 2013-04-03 | Qiagen GmbH | Dry composition comprising a control dye |
WO2013053855A1 (en) | 2011-10-11 | 2013-04-18 | Qiagen Gmbh | Sample processing method and sample processing cartridge |
WO2013068107A1 (en) | 2011-11-07 | 2013-05-16 | Qiagen Gmbh | Lysis method and lysis composition |
EP2730653A1 (en) | 2012-11-07 | 2014-05-14 | QIAGEN GmbH | Method for lysing a fixed biological sample |
US10875022B2 (en) | 2007-07-13 | 2020-12-29 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US10900066B2 (en) | 2006-03-24 | 2021-01-26 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US10913061B2 (en) | 2006-03-24 | 2021-02-09 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
US11060082B2 (en) | 2007-07-13 | 2021-07-13 | Handy Lab, Inc. | Polynucleotide capture materials, and systems using same |
US11078523B2 (en) | 2003-07-31 | 2021-08-03 | Handylab, Inc. | Processing particle-containing samples |
US11141734B2 (en) | 2006-03-24 | 2021-10-12 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US11142785B2 (en) | 2006-03-24 | 2021-10-12 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US11266987B2 (en) | 2007-07-13 | 2022-03-08 | Handylab, Inc. | Microfluidic cartridge |
US11441171B2 (en) | 2004-05-03 | 2022-09-13 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US11453906B2 (en) | 2011-11-04 | 2022-09-27 | Handylab, Inc. | Multiplexed diagnostic detection apparatus and methods |
US11466263B2 (en) | 2007-07-13 | 2022-10-11 | Handylab, Inc. | Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly |
US11549959B2 (en) | 2007-07-13 | 2023-01-10 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
US11788127B2 (en) | 2011-04-15 | 2023-10-17 | Becton, Dickinson And Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
US11806718B2 (en) | 2006-03-24 | 2023-11-07 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0512334A2 (en) * | 1991-05-02 | 1992-11-11 | F. Hoffmann-La Roche Ag | Methods for detecting a target nucleic acid in a sample |
EP0674009A2 (en) * | 1994-03-14 | 1995-09-27 | Becton, Dickinson and Company | Nucleic acid amplification method and apparatus |
WO1997010056A2 (en) * | 1995-09-12 | 1997-03-20 | Becton Dickinson And Company | Device and method for dna amplification and assay |
EP0834729A2 (en) * | 1996-09-26 | 1998-04-08 | Becton, Dickinson and Company | DNA microwell device and method |
US5861251A (en) * | 1996-10-15 | 1999-01-19 | Bioneer Corporation | Lyophilized reagent for polymerase chain reaction |
-
2001
- 2001-05-23 AU AU2001262483A patent/AU2001262483A1/en not_active Abandoned
- 2001-05-23 EP EP01936610A patent/EP1290223A2/en not_active Withdrawn
- 2001-05-23 WO PCT/GB2001/002265 patent/WO2001092569A2/en not_active Application Discontinuation
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0512334A2 (en) * | 1991-05-02 | 1992-11-11 | F. Hoffmann-La Roche Ag | Methods for detecting a target nucleic acid in a sample |
EP0674009A2 (en) * | 1994-03-14 | 1995-09-27 | Becton, Dickinson and Company | Nucleic acid amplification method and apparatus |
WO1997010056A2 (en) * | 1995-09-12 | 1997-03-20 | Becton Dickinson And Company | Device and method for dna amplification and assay |
EP0834729A2 (en) * | 1996-09-26 | 1998-04-08 | Becton, Dickinson and Company | DNA microwell device and method |
US5861251A (en) * | 1996-10-15 | 1999-01-19 | Bioneer Corporation | Lyophilized reagent for polymerase chain reaction |
Cited By (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1350855A2 (en) * | 2002-04-01 | 2003-10-08 | Agilent Technologies, Inc. - a Delaware corporation - | Array based hybridization assays |
EP1350855A3 (en) * | 2002-04-01 | 2003-11-19 | Agilent Technologies, Inc. - a Delaware corporation - | Array based hybridization assays |
EP1498492A1 (en) * | 2003-07-15 | 2005-01-19 | Lukas Bestmann | Apparatus for the preparation of samples |
WO2005007882A2 (en) * | 2003-07-15 | 2005-01-27 | Dual, Jürg | Sample preparing unit |
WO2005007882A3 (en) * | 2003-07-15 | 2005-05-19 | Lukas Bestmann | Sample preparing unit |
US11078523B2 (en) | 2003-07-31 | 2021-08-03 | Handylab, Inc. | Processing particle-containing samples |
US11441171B2 (en) | 2004-05-03 | 2022-09-13 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US7972838B2 (en) | 2004-06-04 | 2011-07-05 | Abacus Diagnostica Oy | Method for stabilizing assay reagents, reagent container with stabilized assay reagents and use thereof |
JP2008501331A (en) * | 2004-06-04 | 2008-01-24 | アバクス ディアグノスティカ オサケ ユキチュア | Method for stabilizing assay reagent, reagent container containing stabilized assay reagent and use thereof |
WO2005118849A1 (en) * | 2004-06-04 | 2005-12-15 | Abacus Diagnostica Oy | Method for stabilizing assay reagents, reagent container with stabilized assay reagents and use thereof |
US7776530B2 (en) | 2004-06-29 | 2010-08-17 | Wallac Oy | Integrated nucleic acid analysis |
US8252536B2 (en) | 2004-06-29 | 2012-08-28 | Wallac Oy | Integrated nucleic acid analysis |
WO2006000648A1 (en) * | 2004-06-29 | 2006-01-05 | Wallac Oy | Integrated nucleic acid analysis |
US10900066B2 (en) | 2006-03-24 | 2021-01-26 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US11806718B2 (en) | 2006-03-24 | 2023-11-07 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US11666903B2 (en) | 2006-03-24 | 2023-06-06 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using same |
US11142785B2 (en) | 2006-03-24 | 2021-10-12 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US11141734B2 (en) | 2006-03-24 | 2021-10-12 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US11085069B2 (en) | 2006-03-24 | 2021-08-10 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US10913061B2 (en) | 2006-03-24 | 2021-02-09 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
EP2210955A3 (en) * | 2006-05-23 | 2010-09-15 | Molecular Detection, Inc. | Ambient temperature stable kits for molecular diagnostics |
US11466263B2 (en) | 2007-07-13 | 2022-10-11 | Handylab, Inc. | Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly |
US10875022B2 (en) | 2007-07-13 | 2020-12-29 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US11254927B2 (en) | 2007-07-13 | 2022-02-22 | Handylab, Inc. | Polynucleotide capture materials, and systems using same |
US11060082B2 (en) | 2007-07-13 | 2021-07-13 | Handy Lab, Inc. | Polynucleotide capture materials, and systems using same |
US11266987B2 (en) | 2007-07-13 | 2022-03-08 | Handylab, Inc. | Microfluidic cartridge |
US11549959B2 (en) | 2007-07-13 | 2023-01-10 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
WO2012010708A1 (en) * | 2010-07-23 | 2012-01-26 | Aj Innuscreen Gmbh | Method, device and test kit for molecular-biological reactions |
US11788127B2 (en) | 2011-04-15 | 2023-10-17 | Becton, Dickinson And Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
EP2574931A1 (en) | 2011-09-29 | 2013-04-03 | Qiagen GmbH | Dry composition comprising a control dye |
CN104066849A (en) * | 2011-10-11 | 2014-09-24 | 凯杰有限公司 | Sample processing method and sample processing cartridge |
EP3663408A1 (en) | 2011-10-11 | 2020-06-10 | QIAGEN GmbH | Sample processing method and sample processing cartridge |
CN104066849B (en) * | 2011-10-11 | 2017-04-19 | 凯杰有限公司 | Sample processing method and sample processing cartridge |
WO2013053855A1 (en) | 2011-10-11 | 2013-04-18 | Qiagen Gmbh | Sample processing method and sample processing cartridge |
US11453906B2 (en) | 2011-11-04 | 2022-09-27 | Handylab, Inc. | Multiplexed diagnostic detection apparatus and methods |
WO2013068107A1 (en) | 2011-11-07 | 2013-05-16 | Qiagen Gmbh | Lysis method and lysis composition |
EP2730653A1 (en) | 2012-11-07 | 2014-05-14 | QIAGEN GmbH | Method for lysing a fixed biological sample |
WO2014072366A1 (en) | 2012-11-07 | 2014-05-15 | Qiagen Gmbh | Method for lysing a fixed biological sample |
Also Published As
Publication number | Publication date |
---|---|
WO2001092569A3 (en) | 2002-03-28 |
EP1290223A2 (en) | 2003-03-12 |
AU2001262483A1 (en) | 2001-12-11 |
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