WO2012055073A1 - Procédé de fixation de la température d'une réaction en chaîne de la polymérase, et instrument associé - Google Patents

Procédé de fixation de la température d'une réaction en chaîne de la polymérase, et instrument associé Download PDF

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Publication number
WO2012055073A1
WO2012055073A1 PCT/CN2010/001730 CN2010001730W WO2012055073A1 WO 2012055073 A1 WO2012055073 A1 WO 2012055073A1 CN 2010001730 W CN2010001730 W CN 2010001730W WO 2012055073 A1 WO2012055073 A1 WO 2012055073A1
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WO
WIPO (PCT)
Prior art keywords
temperature
polymerase chain
chain reaction
heater
reaction according
Prior art date
Application number
PCT/CN2010/001730
Other languages
English (en)
Chinese (zh)
Inventor
苏城
邓秉华
陈柏桦
李馥君
吴思颖
Original Assignee
瑞基海洋生物科技股份有限公司
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 瑞基海洋生物科技股份有限公司 filed Critical 瑞基海洋生物科技股份有限公司
Priority to CN201080069901.6A priority Critical patent/CN103282496B/zh
Priority to PCT/CN2010/001730 priority patent/WO2012055073A1/fr
Publication of WO2012055073A1 publication Critical patent/WO2012055073A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the present invention relates to polymerase chain reaction (PCR), and more particularly to a method and apparatus for temperature setting of a polymerase chain reaction. current technology
  • PCR Polymerase chain reaction
  • This technique generally consists of 20 to 35 thermal cycles, each cycle consisting of the following three different temperature conditions: DNA denaturation at 95 ° C (Denaturation;), primer adhesion at 55 ° C ( Annealing), and an extension reaction was carried out at 72 °C.
  • the general polymerase chain reaction (PCR) is carried out in a thermal cycle apparatus that adjusts the temperature of a medium (including gas, liquid or solid;) to change the temperature of the entire mixture in the capillary tube. Polymerase chain reaction.
  • a primary object of the present invention is to provide a temperature setting method for a polymerase chain reaction which shortens the time required for a polymerase chain reaction.
  • Another object of the present invention is to provide a temperature setting device for a polymerase chain reaction which is low in cost and has great market potential.
  • the present invention provides a temperature setting method for a polymerase chain reaction, which comprises the following steps: (a) placing a test tube containing a mixed solution and a DNA sample in a closed casing (b) Heat the mixture at the bottom of the tube to a denaturation temperature with a heater.
  • the heating mode of the step (b) is a heat conduction mode.
  • the heating mode of the step (b) is a heat radiation mode.
  • the denaturation temperature is between 90 and 98 °C.
  • the invention provides a temperature setting device for polymerase chain reaction, which is used for setting a test tube
  • the temperature of the mixed liquid is characterized in that the temperature setting means comprises: a sealed casing; a heater located in the closed casing and for heating the mixed liquid located at the bottom of the test tube.
  • the heater heats the mixture at the bottom of the tube to 90 to 98 °C.
  • the heater has at least one receptacle for receiving the bottom of the test tube, and the receptacle has an imaginary axis substantially perpendicular to a horizontal plane.
  • the pocket is recessed inwardly from the side of the heater in a semi-cylindrical shape.
  • the cuvette is recessed from the top surface of the heater to have a cylindrical shape.
  • a heat insulating layer is further disposed on the top surface of the heater, and the heat insulating layer has at least one hollow portion, and the hollow portion is located at a position corresponding to the cavity of the heater.
  • the invention uses a heater to heat the mixture at the bottom of the test tube, so that the mixed liquid in the test tube generates heat convection due to the temperature gradient, so that the mixed liquid continuously changes the temperature during the convection process, and the polymerase chain reaction can be convected with the mixture. Loop through. Thereby, the time required for the conventional method to change the temperature can be greatly shortened. Moreover, the temperature setting device using the method has a simple structure and low cost, and thus has great market potential.
  • Figure 1 is a cross-sectional view showing a first preferred embodiment of the present invention
  • Figure 2 is a partial perspective view of a first preferred embodiment of the present invention
  • Figure 3 is a partial perspective view of a second preferred embodiment of the present invention.
  • the temperature setting device 10 for polymerase chain reaction provided by the first preferred embodiment of the present invention is used for setting the temperature of the mixed liquid in a plurality of test tubes T, and setting the temperature.
  • the device 10 is comprised of a hermetic housing 20, a heater 30, and a thermal insulation layer 40.
  • the sealed casing 20 has an upper cover 21, a lower cover 23, and an accommodation space 25 defined by the upper cover 21 and the lower cover 23 for accommodating a plurality of test tubes T.
  • the sealed casing 20 can shield the outside airflow from the outside airflow and affect the temperature inside the sealed casing 20.
  • the heater 30 is disposed in the hermetic casing 20 and is made of a metal having good thermal conductivity.
  • the heater 30 has a plurality of semi-cylindrical cavities 31 recessed inwardly from the side of the heater 30 to accommodate the respective test tubes.
  • the bottom portion, and each of the pockets 31 has an imaginary axis 32 that is perpendicular or approximately perpendicular to a horizontal plane.
  • Heating The device 30 can be set to have a temperature of about 90 to 98 ° C as needed, and heat the mixture located at the bottom of the test tube T to a denaturing temperature by heat conduction.
  • the heat insulating layer 40 covers the top surface of the heater 30, and the heat insulating layer 40 has a plurality of hollow portions 41 corresponding to the respective pockets 31 of the heater 30 for the test tube T to penetrate.
  • the heat insulating layer 40 blocks the heat of the heater 30 so that the temperature of the mixture in the test tube T above the heat insulating layer 40 is not affected by the heater 30.
  • a plurality of test tubes T containing the mixture and the DNA sample can be placed on a holder C (this is a conventional technique, which is not described above), so that each tube T passes through the heat insulation.
  • the hollow portion 41 of the layer 40 has its bottom end accommodated in the cavity 31 of the heater 30, and the heater 30 can heat the mixture at the bottom of the test tube T to a denaturation temperature, usually 90 to 98 ° C, in this embodiment. In the example, it is about 95 ° C.
  • the mixture in the test tube T generates heat convection due to the temperature gradient, causing the mixture at the bottom to flow upward and gradually cool down.
  • the liquid level of the mixed liquid in the test tube ⁇ is approximately equal to room temperature (about 25 ° C), and the mixture starts to heat due to the temperature gradient as the mixture at the bottom of the test tube T is heated. Convection, the liquid level of the mixed liquid will also gradually rise.
  • the mixed liquid in the test tube T will have a bonding temperature just at the primer (for example, 55 ° C;), where the primer can be bonded, and
  • the mixture in the test tube T also has another temperature (about 72 ° C) in which the primer is subjected to the extension reaction due to the temperature gradient, where the extension reaction can be carried out while the mixture continues to convect downward to the bottom of the test tube T.
  • the denaturation reaction can be carried out again, and the cycle is repeated to achieve rapid replication of specific DNA fragments for detection purposes.
  • the liquid level of the mixture will gradually rise above 55 °C, exceeding the bonding temperature of the primer, but in fact, the mixture convection cycle before the temperature rises to 55 °C.
  • the number of times is sufficient to complete the polymerase chain reaction. Thereby, the time required for the conventional method to change the temperature can be greatly shortened, and the temperature setting device using the method has a simple structure, low manufacturing cost, and great market potential.
  • the aforementioned mixture contains a buffer solution, a base pair (; dNTPs), a polymerization ta per polymer (Taq polymerase), and a primer.
  • the structure of the heater in the foregoing embodiment can be varied.
  • a temperature setting device according to a second preferred embodiment of the present invention, wherein the receptacle 51 of the heater 50 is recessed from the top surface of the heater 50 to have a cylindrical shape.
  • the heating method of the heater may be changed to heat the mixed liquid by heat radiation as needed, for example, by microwave heating.
  • the number of tanks of the heater can be set one or one as needed.
  • the number of the hollow portions of the heat insulating layer corresponding thereto is also the same.

Abstract

L'invention concerne un procédé de fixation de la température d'une réaction en chaîne de la polymérase, comprenant la mise en place d'un tube ayant une liqueur mixte et un échantillon d'ADN dans une enveloppe étanche à l'air, et le chauffage de la liqueur mixte située au fond du tube jusqu'à une température de dénaturation au moyen d'un dispositif de chauffage. Une convection thermique peut avoir lieu dans la liqueur mixte à cause du gradient de température et modifie la température de la liqueur mixte au cours du procédé de convection, ainsi la réaction en chaîne de la polymérase peut avoir lieu de manière circulaire avec la convection de la liqueur mixte. À l'aide du procédé de la présente invention, la durée de la réaction en chaîne de la polymérase peut être raccourcie et le coût de l'instrument de fixation de la température est faible.
PCT/CN2010/001730 2010-10-29 2010-10-29 Procédé de fixation de la température d'une réaction en chaîne de la polymérase, et instrument associé WO2012055073A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201080069901.6A CN103282496B (zh) 2010-10-29 2010-10-29 聚合酶连锁反应的温度设定方法及装置
PCT/CN2010/001730 WO2012055073A1 (fr) 2010-10-29 2010-10-29 Procédé de fixation de la température d'une réaction en chaîne de la polymérase, et instrument associé

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2010/001730 WO2012055073A1 (fr) 2010-10-29 2010-10-29 Procédé de fixation de la température d'une réaction en chaîne de la polymérase, et instrument associé

Publications (1)

Publication Number Publication Date
WO2012055073A1 true WO2012055073A1 (fr) 2012-05-03

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PCT/CN2010/001730 WO2012055073A1 (fr) 2010-10-29 2010-10-29 Procédé de fixation de la température d'une réaction en chaîne de la polymérase, et instrument associé

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Country Link
CN (1) CN103282496B (fr)
WO (1) WO2012055073A1 (fr)

Cited By (1)

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US9505003B2 (en) 2014-11-14 2016-11-29 Industrial Technology Research Institute Portable real-time heating and detection device

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* Cited by examiner, † Cited by third party
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TWI567189B (zh) * 2015-09-21 2017-01-21 Biochemical reactor
CN105219637B (zh) * 2015-09-25 2017-07-21 瑞基海洋生物科技股份有限公司 生化反应器

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CN1680574A (zh) * 2004-03-12 2005-10-12 三星电子株式会社 用于扩增核酸的方法及设备
CN1690218A (zh) * 2004-04-26 2005-11-02 佳能株式会社 Pcr扩增反应装置、以及利用该装置的pcr扩增反应方法
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WO2009094638A2 (fr) * 2008-01-24 2009-07-30 Medigen Biotechnology Corp. Procédés et appareils pour une réaction en chaîne par polymérase (pcr) convective

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CN1680574A (zh) * 2004-03-12 2005-10-12 三星电子株式会社 用于扩增核酸的方法及设备
CN1690218A (zh) * 2004-04-26 2005-11-02 佳能株式会社 Pcr扩增反应装置、以及利用该装置的pcr扩增反应方法
CN101200694A (zh) * 2007-10-25 2008-06-18 刘文韬 闭环式聚合酶链反应系统及其制造方法
WO2009094638A2 (fr) * 2008-01-24 2009-07-30 Medigen Biotechnology Corp. Procédés et appareils pour une réaction en chaîne par polymérase (pcr) convective

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9505003B2 (en) 2014-11-14 2016-11-29 Industrial Technology Research Institute Portable real-time heating and detection device

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CN103282496A (zh) 2013-09-04
CN103282496B (zh) 2015-04-22

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