EP1290223A2 - Formulation pour amplification en chaine par polymerase et recipient contenant celle-ci - Google Patents

Formulation pour amplification en chaine par polymerase et recipient contenant celle-ci

Info

Publication number
EP1290223A2
EP1290223A2 EP01936610A EP01936610A EP1290223A2 EP 1290223 A2 EP1290223 A2 EP 1290223A2 EP 01936610 A EP01936610 A EP 01936610A EP 01936610 A EP01936610 A EP 01936610A EP 1290223 A2 EP1290223 A2 EP 1290223A2
Authority
EP
European Patent Office
Prior art keywords
formulation
vessel
polymerase
chain reaction
effecting
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.)
Withdrawn
Application number
EP01936610A
Other languages
German (de)
English (en)
Inventor
John Douglas Oultram
Conor Joseph Mulrooney
Jacqueline Clare Coutts
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.)
Tepnel Medical Ltd
Original Assignee
Tepnel Medical Ltd
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
Priority claimed from GB0013043A external-priority patent/GB0013043D0/en
Priority claimed from GB0013863A external-priority patent/GB0013863D0/en
Application filed by Tepnel Medical Ltd filed Critical Tepnel Medical Ltd
Publication of EP1290223A2 publication Critical patent/EP1290223A2/fr
Withdrawn legal-status Critical Current

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
  • the PCR reaction itself generally consists of a number of preparative steps including the addition of a buffer solution, dNTP mix, primer solutions and usually a separate MgCl 2 solution followed by the addition of target and DNA polymerase.
  • Many of the reagents can be included in a "master mix” that is then dispensed singly to individual reactions.
  • Other reagents, usually the target and polymerase enzyme must be added individually to reaction tubes, which involves pipetting very low volumes (sub to low ⁇ L) which can lead to considerable reaction variability.
  • the complexity of the steps involved in optimising the performing PCR in this fashion requires a high degree of expertise in those entrusted with its performance and constant vigilance and monitoring of contamination issues.
  • 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.
  • Hot-start polymerases are known in the art and are such that a heating step is required to activate the polymerase (which has typically been inactivated with an antibody).
  • the "hot-start” polymerase should be one for which the "means” of inactivation of the enzyme (e.g. an antibody) must be able to withstand the drying/rehydration procedure.
  • the advantage that the use of such an enzyme confers is that the re-hydration of the dried reagent composition can occur at ambient temperature without initiating potentially ruinous side reactions prior to heating the sample and cooling to annealing temperature at which only desired reactions can occur.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une formulation s'utilisant pour mettre en oeuvre une amplification en chaîne par polymérase, une composition séchée de réactifs renfermant un tampon de réaction, des dNTP, au moins deux amorces et une polymérase. La formulation est réhydratable de manière à permettre l'amplification d'une séquence d'acides nucléiques voulue. La formulation incorpore une molécule rapporteur fluorescente capable de rapporter par détection homologue la présence d'acide nucléique amplifié, produit par l'amplification en chaîne par polymérase.
EP01936610A 2000-05-31 2001-05-23 Formulation pour amplification en chaine par polymerase et recipient contenant celle-ci Withdrawn EP1290223A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB0013043 2000-05-31
GB0013043A GB0013043D0 (en) 2000-05-31 2000-05-31 Formulation for polymerase chain reaction and vessel containing same
GB0013863 2000-06-07
GB0013863A GB0013863D0 (en) 2000-06-07 2000-06-07 Formulation for polymerase chain reaction and vessel containing same
PCT/GB2001/002265 WO2001092569A2 (fr) 2000-05-31 2001-05-23 Formulation pour amplification en chaine par polymerase et recipient contenant celle-ci

Publications (1)

Publication Number Publication Date
EP1290223A2 true EP1290223A2 (fr) 2003-03-12

Family

ID=26244374

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01936610A Withdrawn EP1290223A2 (fr) 2000-05-31 2001-05-23 Formulation pour amplification en chaine par polymerase et recipient contenant celle-ci

Country Status (3)

Country Link
EP (1) EP1290223A2 (fr)
AU (1) AU2001262483A1 (fr)
WO (1) WO2001092569A2 (fr)

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US20030186252A1 (en) * 2002-04-01 2003-10-02 Ilsley Diane D. Array based hybridization assays employing enzymatically generated labeled target nucleic acids and compositions for practicing the same
EP1498492B1 (fr) * 2003-07-15 2011-11-02 Lukas Bestmann Appareil pour la preparation des echantillons
EP2402089A1 (fr) 2003-07-31 2012-01-04 Handylab, Inc. Traitement d'échantillons contenant des particules
US8852862B2 (en) 2004-05-03 2014-10-07 Handylab, Inc. Method for processing polynucleotide-containing samples
FI20040768A0 (fi) 2004-06-04 2004-06-04 Teemu Korpimaeki Menetelmä määritysreagenssien stabiloimiseksi, stabilisoituja määritysreagensseja sisältävä reagenssisäiliö ja sen käyttö
FI20045248A (fi) * 2004-06-29 2005-12-30 Wallac Oy Integroitu nukleiinihappoanalyysi
US7776530B2 (en) 2004-06-29 2010-08-17 Wallac Oy Integrated nucleic acid analysis
US7998708B2 (en) 2006-03-24 2011-08-16 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
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
ES2692380T3 (es) 2006-03-24 2018-12-03 Handylab, Inc. Método para realizar PCR con un cartucho con varias pistas
US8883490B2 (en) 2006-03-24 2014-11-11 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US20080050737A1 (en) * 2006-05-23 2008-02-28 Boaz Arieli Ambient Temperature Stable Kits for Molecular Diagnostics
WO2008061165A2 (fr) 2006-11-14 2008-05-22 Handylab, Inc. Cartouche microfluidique et son procédé de fabrication
US9186677B2 (en) 2007-07-13 2015-11-17 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
WO2009012185A1 (fr) 2007-07-13 2009-01-22 Handylab, Inc. Matières absorbant les polynucléotides, et procédés d'utilisation de celles-ci
US8287820B2 (en) 2007-07-13 2012-10-16 Handylab, Inc. Automated pipetting apparatus having a combined liquid pump and pipette head system
US8182763B2 (en) 2007-07-13 2012-05-22 Handylab, Inc. Rack for sample tubes and reagent holders
US8105783B2 (en) 2007-07-13 2012-01-31 Handylab, Inc. Microfluidic cartridge
DE102010038330A1 (de) * 2010-07-23 2012-03-01 Aj Innuscreen Gmbh Verfahren, Vorrichtung und Testkit für Molekularbiologische Reaktionen
CN106148512B (zh) 2011-04-15 2020-07-10 贝克顿·迪金森公司 扫描实时微流体热循环仪和用于同步的热循环和扫描光学检测的方法
EP2574931B1 (fr) 2011-09-29 2017-03-22 Qiagen GmbH Composition sèche comprenant un colorant de contrôle
JP6262657B2 (ja) 2011-10-11 2018-01-17 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 試料プロセシング方法および試料プロセシングカートリッジ
CN104040238B (zh) 2011-11-04 2017-06-27 汉迪拉布公司 多核苷酸样品制备装置
EP2776577B1 (fr) 2011-11-07 2017-01-11 Qiagen GmbH Procédé et composition de lyse
EP2730653A1 (fr) 2012-11-07 2014-05-14 QIAGEN GmbH Procédé de lyse d'un échantillon biologique fixe

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US5994056A (en) * 1991-05-02 1999-11-30 Roche Molecular Systems, Inc. Homogeneous methods for nucleic acid amplification and detection
CA2143365A1 (fr) * 1994-03-14 1995-09-15 Hugh V. Cottingham Methode et appareil pour l'amplification de l'acide nucleique
AU7238396A (en) * 1995-09-12 1997-04-01 Becton Dickinson & Company Device and method for dna amplification and assay
US5795748A (en) * 1996-09-26 1998-08-18 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

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Also Published As

Publication number Publication date
WO2001092569A3 (fr) 2002-03-28
AU2001262483A1 (en) 2001-12-11
WO2001092569A2 (fr) 2001-12-06

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