WO2001081619A2 - Conductivity pcr - Google Patents

Conductivity pcr Download PDF

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Publication number
WO2001081619A2
WO2001081619A2 PCT/DE2001/001025 DE0101025W WO0181619A2 WO 2001081619 A2 WO2001081619 A2 WO 2001081619A2 DE 0101025 W DE0101025 W DE 0101025W WO 0181619 A2 WO0181619 A2 WO 0181619A2
Authority
WO
WIPO (PCT)
Prior art keywords
conductivity
pcr
reaction
thermal cycler
polymerase chain
Prior art date
Application number
PCT/DE2001/001025
Other languages
German (de)
French (fr)
Other versions
WO2001081619A8 (en
WO2001081619A3 (en
Inventor
Borros Arneth
Alfons Arneth
Original Assignee
Borros Arneth
Alfons Arneth
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 DE20007376U external-priority patent/DE20007376U1/en
Priority claimed from DE20007378U external-priority patent/DE20007378U1/en
Priority claimed from DE20013567U external-priority patent/DE20013567U1/en
Priority claimed from DE20018005U external-priority patent/DE20018005U1/en
Application filed by Borros Arneth, Alfons Arneth filed Critical Borros Arneth
Priority to AU56115/01A priority Critical patent/AU5611501A/en
Publication of WO2001081619A2 publication Critical patent/WO2001081619A2/en
Publication of WO2001081619A3 publication Critical patent/WO2001081619A3/en
Publication of WO2001081619A8 publication Critical patent/WO2001081619A8/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
    • G01N27/06—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors

Definitions

  • CONDUCTIVITY PCR (conductivity PCR)
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • Previously customary "online" PCR apparatus either stained the DNA formed in the course of the PCR or used fluorescent-labeled oligonucleotide primers to quantify the course of the PCR. A complex optical system for measuring the respective fluorescence was required.
  • This invention is based on the consideration of using the amount of phosphate which arises when the PCR reaction proceeds and the associated changes in conductivity as markers for the amplification factor of the PCR reaction. During the course of a PCR reaction, this apparatus determines the phosphate increase “online” by means of small microelectrodes which are immersed in the PCR solution.
  • the apparatus is able to measure the amount of DNA or RNA before the PCR reaction
  • the amount of phosphate is selected as a marker for detection on the basis of its good specific electrical conductivity and on the basis of its rise in the course of the annealing and extension phase of the PCR, and a microprocessor can then determine the phosphate concentration after the end using a previously determined calibration curve and the determined measured values of the PCR reaction, the amplification factor of the PCR reaction and the DNA or RNA concentration before the start of the PCR reaction, using the time course of the increase in conductivity as a function of the number of cycles and the temperature to determine the original concentration of DNA ,
  • An advantage of this invention is that an electronically usable measure is used directly with the conductivity.
  • the amount of phosphate is also a particularly sensitive parameter.
  • the conductivity changes within each cycle depending on the temperature in accordance with the three phases (annealing, extension, denaturation). Overall, the conductivity decreases in the course of the PCR. In the annealing phase, the conductivity decreases relatively strongly. In the first approximately ten cycles, the conductivity increases linearly ("linear phase") up to the "trashhold cycle". This cycle is particularly suitable for quantifying the polymerase chain reaction. The exponential phase begins from the Trashhold cycle. This is characterized by a total decrease in conductivity. Nevertheless, a phase-wise increase in conductivity remains during the denaturation phase and the extension phase. The changes in conductivity of the individual phases are also suitable for PCR quantification.

Abstract

The invention is based on the concept of using the change in conductivity to be measured during the PCR as a marker for the progression of the PCR and for the quantification of the starting quantity of DNA. This apparatus determines the changes in conductivity 'online' during the course of a PCR by means of small microelectrodes (4) which are immersed in the PCR solution. Using this parameter, the apparatus is able to calculate the quantity of DNA or RNA before the conclusion of the PCR.

Description

Beschreibung:Description:
KONDUKTIVITÄTS-PCR (Leitfähigkeits PCR)CONDUCTIVITY PCR (conductivity PCR)
Eine in der Molekularbiologie wichtige Fragestellung ist die nach der Aktivität einzelner Gene. Um einzelne Gene in ihrer Aktivität zu bestimmen wird häufig eine PCR (Polymerasekettenreaktion) oder eine RT-PCR (reverse Transkriptase Polymerase Kettenreaktion) durchgeführt. Eine Schwierigkeit besteht darin, daß die PCR-Reaktion nur schwierig quantifizierbar ist, daß heißt die Menge an DNA/RNA vor Ablauf der PCR-Reaktion läßt sich nur abschätzen.An important question in molecular biology is that of the activity of individual genes. To determine individual genes in their activity, a PCR (polymerase chain reaction) or an RT-PCR (reverse transcriptase polymerase chain reaction) is often carried out. One difficulty is that the PCR reaction is difficult to quantify, that is, the amount of DNA / RNA before the PCR reaction is complete can only be estimated.
Bisher übliche Apparate zur „online" PCR färben entweder die im Verlauf der PCR gebildete DNA oder verwenden floureszenzmarkierte Oligonucleotidprimer um den Verlauf der PCR zu quantifizieren. Dabei ist ein aufwendiges optisches System zur Messung der jeweiligen Fluoreszenzen nötig.Previously customary "online" PCR apparatus either stained the DNA formed in the course of the PCR or used fluorescent-labeled oligonucleotide primers to quantify the course of the PCR. A complex optical system for measuring the respective fluorescence was required.
Dieser Erfindung liegt die Überlegung zugrunde die bei Ablauf der PCR-Reaktion entstehende Menge an Phosphat und die damit verbundene Leitfähigkeitsänderungen als Marker für den Verstärkungsfaktor der PCR-Reaktion zu nutzen. Dieser Apparat bestimmt während dem Ablauf einer PCR-Reaktion „online" mittels kleiner Mikroelektroden, die in die PCR-Lösung eintauchen, den Phosphatanstieg. Aufgrund dieses Parameters ist der Apparat in der Lage die DNA bzw. RNA- Menge vor Ablauf der PCR-Reaktion zu errechnen. Die Phosphatmenge wird dabei aufgrund ihrer guten spezifischen elektrischen Leitfähigkeit und aufgrund ihres Anstiegs im Verlauf der Annealing und Extension- phase der PCR als Marker zur Detektion gewählt. Anhand einer zuvor ermittelten Eichkurve und der bestimmten Meßwerte kann anschließend ein Mikroprozessor die Phosphatkonzentration nach Ablauf der PCR-Reaktion, den Verstärkungsfaktor der PCR-Reaktion sowie die DNA- bzw. die RNA-Konzentration vor Beginn der PCR- Reaktion errechnen. Dabei eignet sich der zeitliche Verlauf des Leitfähigkeitsanstiegs als Funktion der Zyklenzahl und der Temperatur zur Ermittlung der Ursprungskonzentration an DNA.This invention is based on the consideration of using the amount of phosphate which arises when the PCR reaction proceeds and the associated changes in conductivity as markers for the amplification factor of the PCR reaction. During the course of a PCR reaction, this apparatus determines the phosphate increase “online” by means of small microelectrodes which are immersed in the PCR solution. Because of this parameter, the apparatus is able to measure the amount of DNA or RNA before the PCR reaction The amount of phosphate is selected as a marker for detection on the basis of its good specific electrical conductivity and on the basis of its rise in the course of the annealing and extension phase of the PCR, and a microprocessor can then determine the phosphate concentration after the end using a previously determined calibration curve and the determined measured values of the PCR reaction, the amplification factor of the PCR reaction and the DNA or RNA concentration before the start of the PCR reaction, using the time course of the increase in conductivity as a function of the number of cycles and the temperature to determine the original concentration of DNA ,
Vorteilhaft an dieser Erfindung ist, daß mit der Leitfähigkeit direkt ein elektronisch verwertbares Maß verwendet wird. Zudem ist die Phosphatmenge ein besonders empfindlicher Parameter. Die Leitfähigkeit ändert sich dabei innerhalb eines jeden Zyklus temperaturabhängig entsprechend den drei Phasen (Annealing, Extension, Denaturierung). Insgesamt nimmt die Leitfähigkeit dabei im Verlauf der PCR ab. In der Annealingphase nimmt die Leitfähigkeit relativ stark ab. In den ersten circa zehn Zyklen steigt die Leitfähigkeit linear („lineare Phase") bis zum „Trashhold Zyklus". Dieser Zyklus eignet sich in besonderer Weise zur Quantifizierung der Polymerasekettenreaktion. Ab dem Trashhold Zyklus beginnt die exponentielle Phase. Diese ist durch eine summarische Leitfähigkeitsabnahme gekennzeichnet. Dennoch bleibt auch hier wähend der Denaturierungsphase und der Extensionsphase eine phasenweise Leitfähigkeitszunahme erhalten. Die Leitfähigkeitsänderungen der einzelnen Phasen eignen sich ebenfalls zur PCR- Quantifizierung.An advantage of this invention is that an electronically usable measure is used directly with the conductivity. The amount of phosphate is also a particularly sensitive parameter. The conductivity changes within each cycle depending on the temperature in accordance with the three phases (annealing, extension, denaturation). Overall, the conductivity decreases in the course of the PCR. In the annealing phase, the conductivity decreases relatively strongly. In the first approximately ten cycles, the conductivity increases linearly ("linear phase") up to the "trashhold cycle". This cycle is particularly suitable for quantifying the polymerase chain reaction. The exponential phase begins from the Trashhold cycle. This is characterized by a total decrease in conductivity. Nevertheless, a phase-wise increase in conductivity remains during the denaturation phase and the extension phase. The changes in conductivity of the individual phases are also suitable for PCR quantification.
Möglicherweise binden bei der Annealingtemperatur viele Mononucleotide und viele Magnesiumionen sowie andere Ionen an die DNA. Mit Beginn der Extensionsphase steigt die Leitfähigkeit kontinuierlich bis zum Ende der Extensionsphase an. Dieser Anstieg wird vermutlich durch die Phosphatfreisetzung bewirkt. Und muß gemessen werden. An die Elongationsphase schließt sich die Denaturierung an, hier steigt die Leitfähigkeit auf ein Maximum. Vermutlich dissoziieren in dieser Phase alle Ionen ab. Erläuterung anhand eines Ausführungsbeispiels: ZeichnungPossibly many mononucleotides and many magnesium ions and other ions bind to the DNA at the annealing temperature. With the beginning of the extension phase, the conductivity increases continuously until the end of the extension phase. This increase is thought to be caused by the release of phosphate. And must be measured. The denaturation follows the elongation phase, here the conductivity increases to a maximum. Presumably, all ions dissociate in this phase. Explanation based on an embodiment: drawing
1) Thermocycler1) Thermal cycler
2) PCR-Reaktionsgefäß2) PCR reaction tube
3) PCR-Reaktionslösung3) PCR reaction solution
4) Mikroelektroden zur Leitfähigkeitsmessung4) Microelectrodes for conductivity measurement
5) Mikroprozessor zur Registrierung der Leitfähigkeitsmeßwerte5) Microprocessor for registering the conductivity measurements
6) Kontakte 6) contacts

Claims

Konduktivitäts-PCRPatentansprücheUnabhängiger Hauptanspruch: Conductivity PCR Patent Claims Main Independent Claim:
1.) Ein Apparat zur Durchführung einer PCR-Reaktion (Polymerasekettenreaktion) in einem sogenannten "Thermocycler", dadurch gekennzeichnet, daß dieser Apparat „online" während dem Ablauf einer PCR-Reaktion mittels kleiner in die PCR-Lösung eintauchender Mikroelektroden die Änderung der elektrischen Leitfähigkeit der Lösung kontinuierlich oder diskontinuierich (z.B. nur während der Annealing/Extension-phase oder nur während der ersten Zyklen) verfolgt.1.) An apparatus for carrying out a PCR reaction (polymerase chain reaction) in a so-called "thermal cycler", characterized in that this apparatus "online" during the course of a PCR reaction by means of small microelectrodes immersed in the PCR solution the change in the electrical The conductivity of the solution is monitored continuously or discontinuously (for example only during the annealing / extension phase or only during the first cycles).
Abhängige Nebenansprüche:Dependent subsidiary claims:
2.) Ein Thermocycler nach Schutzanspruch 1) dadurch gekennzeichnet, daß dieser Thermocycler den Übergang des linearen Leitfähigkeitsanstiegs zum ersten Leitfähigkeitsabfall als trash-hold cycle zur Quantifizierung nutzt.2.) A thermal cycler according to protection claim 1), characterized in that this thermal cycler uses the transition from the linear increase in conductivity to the first decrease in conductivity as a trash-hold cycle for quantification.
3.) Reaktionsgefäße zur Durchführung einer Polymerasekettenreaktion, dadurch gekennzeichnet, daß Mikroelektroden in die verwendeten Reaktionsgefäße integriert sind.3.) reaction vessels for carrying out a polymerase chain reaction, characterized in that microelectrodes are integrated in the reaction vessels used.
4.) Eine Multi well Platte zur Durchführung einer Polymerasekettenreaktion, dadurch gekennzeichnet, daß Mikroelektroden in die einzelnen Wells der multi well Platte integriert sind.4.) A multi well plate for carrying out a polymerase chain reaction, characterized in that microelectrodes are integrated in the individual wells of the multi well plate.
5.)Ein Thermocycler nach Schutzanspruch 1-4, zusätzlich dadurch gekennzeichnet, daß neben der elektrischen Leitfähigkeit auch noch die aktuelle Temperatur in der5.) A thermal cycler according to protection claims 1-4, additionally characterized in that in addition to the electrical conductivity, the current temperature in the
Reaktionslösung gemessen wird, daß die verschiedenen bekannten Möglichkeiten zur Heizung / Kühlung (z.B. Luftheizung / Kühlung oder Peltierelemente) Anwendung finden und das Kontakte zur Verwendung der Reaktionsgefäße nachReaction solution is measured that the various known heating / cooling options (e.g. air heating / cooling or Peltier elements) are used and the contacts for using the reaction vessels
Schutzanspruch 3 und 4 vorhanden sind.Protection claims 3 and 4 are available.
6.)Eine elektrische Apperatur, dadurch gekennzeichnet, daß diese die im Verlauf der Polymerasekettenreaktion gemessenen Parameter (es sind dies die elektrische Leitfähigkeit, Temperatur, Zykluszahl und Zeit) registriert und sinnvoll verknüpft.6.) An electrical apparatus, characterized in that it registers and sensibly combines the parameters measured in the course of the polymerase chain reaction (these are the electrical conductivity, temperature, number of cycles and time).
7.) Ein Thermocycler nach Schutzanspruch 1-4 mit der Möglichkeit zur abschließenden Schmelzkurvenanalyse, zusätzlich dadurch gekennzeichnet, daß während der Schmelzkurvenanalyse die elektrische Leitfähigkeit gemessen wird. 7.) A thermal cycler according to protection claims 1-4 with the possibility of a final melting curve analysis, additionally characterized in that the electrical conductivity is measured during the melting curve analysis.
PCT/DE2001/001025 2000-04-22 2001-03-17 Conductivity pcr WO2001081619A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56115/01A AU5611501A (en) 2000-04-22 2001-03-17 Conductivity pcr

Applications Claiming Priority (16)

Application Number Priority Date Filing Date Title
DE20007376U DE20007376U1 (en) 2000-04-22 2000-04-22 PCR conductivity microarrays
DE20007378.8 2000-04-22
DE20007378U DE20007378U1 (en) 2000-04-22 2000-04-22 Phosphate conductivity PCR
DE20007376.1 2000-04-22
US20419200P 2000-05-08 2000-05-08
US60/204,192 2000-05-08
US21942100P 2000-07-20 2000-07-20
US21942200P 2000-07-20 2000-07-20
US60/219422 2000-07-20
US60/219,421 2000-07-20
DE20013567U DE20013567U1 (en) 2000-08-08 2000-08-08 Conductivity PCR
DE20013567.8 2000-08-08
DE20018005.3 2000-10-22
DE20018005U DE20018005U1 (en) 2000-10-22 2000-10-22 Sandwich PCR DNA microarray
DE10060256.8 2000-12-04
DE10060256 2000-12-04

Publications (3)

Publication Number Publication Date
WO2001081619A2 true WO2001081619A2 (en) 2001-11-01
WO2001081619A3 WO2001081619A3 (en) 2002-05-16
WO2001081619A8 WO2001081619A8 (en) 2002-06-13

Family

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PCT/DE2001/001025 WO2001081619A2 (en) 2000-04-22 2001-03-17 Conductivity pcr

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WO (1) WO2001081619A2 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1420070A1 (en) * 2002-11-12 2004-05-19 Samsung Electronics Co., Ltd. Method for detecting PCR products from electrical signal generation
WO2010089470A1 (en) * 2009-02-06 2010-08-12 Bio-Rad Pasteur Thermal validation apparatus, assembly including a device for the thermal processing of biological samples and such an apparatus, and method for manufacturing such an apparatus
WO2013150454A2 (en) * 2012-04-04 2013-10-10 Shama Bhat System and method for detecting and analyzing amplified deoxyribonucleic acid
DE102013010961A1 (en) 2013-07-01 2015-01-08 Borros Arneth DNA sequencing by conductivity measurement of the DNA polymerase

Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0488769A2 (en) * 1990-11-29 1992-06-03 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
WO1999010530A1 (en) * 1997-08-22 1999-03-04 Molecular Sensors Limited Estimation of nucleic acid
US5891639A (en) * 1993-11-12 1999-04-06 Geron Corporation Telomerase activity assays

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0488769A2 (en) * 1990-11-29 1992-06-03 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5891639A (en) * 1993-11-12 1999-04-06 Geron Corporation Telomerase activity assays
WO1999010530A1 (en) * 1997-08-22 1999-03-04 Molecular Sensors Limited Estimation of nucleic acid

Non-Patent Citations (1)

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Title
J. P. SCHONFIELD: "a rapid semi-automated microtiter plate method for analysis and sequencing by PCR from bacterial stocks" NUCLEIC ACIDS RESEARCH, Bd. 17, Nr. 22, 1989, Seite 9498 XP001053732 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1420070A1 (en) * 2002-11-12 2004-05-19 Samsung Electronics Co., Ltd. Method for detecting PCR products from electrical signal generation
JP2004159657A (en) * 2002-11-12 2004-06-10 Samsung Electronics Co Ltd Method for detecting pcr product by using electrical signal
US7135294B2 (en) 2002-11-12 2006-11-14 Samsung Electronics Co., Ltd. Method for detecting PCR product using electrical signal
KR100858080B1 (en) * 2002-11-12 2008-09-10 삼성전자주식회사 A method for detecting a PCR product by measuring a electrical signal
WO2010089470A1 (en) * 2009-02-06 2010-08-12 Bio-Rad Pasteur Thermal validation apparatus, assembly including a device for the thermal processing of biological samples and such an apparatus, and method for manufacturing such an apparatus
FR2941876A1 (en) * 2009-02-06 2010-08-13 Bio Rad Pasteur THERMAL VALIDATION APPARATUS, ASSEMBLY OF A DEVICE FOR PROCESSING BIOLOGICAL SAMPLES AND SUCH APPARATUS, AND METHOD FOR MANUFACTURING SUCH APPARATUS
JP2012517220A (en) * 2009-02-06 2012-08-02 バイオ−ラッド・イノヴァシオン Thermal verification apparatus, apparatus for heat treatment of biological sample, assembly including the thermal verification apparatus, and method for manufacturing the thermal verification apparatus
AU2009339202B2 (en) * 2009-02-06 2015-04-02 Bio-Rad Europe Gmbh Thermal validation apparatus, assembly including a device for the thermal processing of biological samples and such an apparatus, and method for manufacturing such an apparatus
US9221054B2 (en) 2009-02-06 2015-12-29 Bio-Rad Innovations Thermal validation apparatus, assembly including a device for the thermal processing of biological samples and such an apparatus, and method for manufacturing such an apparatus
WO2013150454A2 (en) * 2012-04-04 2013-10-10 Shama Bhat System and method for detecting and analyzing amplified deoxyribonucleic acid
WO2013150454A3 (en) * 2012-04-04 2013-11-28 Shama Bhat System and method for detecting and analyzing amplified deoxyribonucleic acid
DE102013010961A1 (en) 2013-07-01 2015-01-08 Borros Arneth DNA sequencing by conductivity measurement of the DNA polymerase

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Publication number Publication date
WO2001081619A8 (en) 2002-06-13
WO2001081619A3 (en) 2002-05-16

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