EP0990050A1 - Evaluation d'un acide nucleique - Google Patents

Evaluation d'un acide nucleique

Info

Publication number
EP0990050A1
EP0990050A1 EP98939776A EP98939776A EP0990050A1 EP 0990050 A1 EP0990050 A1 EP 0990050A1 EP 98939776 A EP98939776 A EP 98939776A EP 98939776 A EP98939776 A EP 98939776A EP 0990050 A1 EP0990050 A1 EP 0990050A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
conductivity
parameter
molecular weight
property
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Withdrawn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
EP98939776A
Other languages
German (de)
English (en)
Inventor
Benjamin David Cobb
John Michael Clarkson
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.)
Molecular Sensors Ltd
Original Assignee
Molecular Sensors 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
Application filed by Molecular Sensors Ltd filed Critical Molecular Sensors Ltd
Publication of EP0990050A1 publication Critical patent/EP0990050A1/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • This invention relates to the estimation of nucleic acid and more particularly to the estimation of a property or parameter of a nucleic acid or of a process in which a nucleic acid is modified by chemical or biochemical reaction.
  • the invention is especially directed to the determination of concentration (quantification) of nucleic acids in solution and to the determination of molecular weight (sizing) of nucleic acid molecules.
  • the invention is also applicable to the monitoring of reactions in which nucleic acids are modified chemically or biochemically.
  • DNA concentra ion is determined spectrophotometrically, a method which relies on the characteristic absorption of ultra-violet light (ca. 260nm) by the nucleotide ring structure of DNA molecules.
  • DNA molecules are normally size-separated by agarose gel electrophoresis and then visualised using the dye ethidium bromide.
  • DNA can also be quantified in agarose gels by comparing with known standards by measuring the fluorescence emitted following excitation by ultra-violet light (ca. 300nm) . Both methods have inherent disadvantages.
  • UV spectrophotometers are expensive pieces of equipment requiring the use of costly quartz curvettes and rely on the 'destructive' processing of relatively large sample volumes .
  • ethidium bromide for DNA visualisation and quantification, although a cheaper alternative, is also undesirable due to its extremely toxic and carcinogenic nature .
  • the present invention comprises a method for the estimation of a property or parameter of a nucleic acid material, or of a process in which a nucleic acid is modified bv a chemical or biochemical reaction, said property or parameter being one to which the electrical conductivity of the nucleic acid material is related, which method comprises measuring the electrical conductivity of the nucleic acid material, or of a reaction mixture containing said material, and estimating from said measurement the property or parameter of the material or process by reference to a predetermined relationship between electrical conductivity and said property or parameter.
  • the present invention is based on the discovery that there are certain important properties of nucleic acids, the quantitative determination of which is frequently desirable, which can be assessed by measurement of the conductivity of a solution of the nucleic acid or acids. Changes in such properties may be reflected in corresponding changes of electrical conductivity.
  • concentration of nucleic acid in solution and the molecular weight of a species of nucleic acid are examples of important property which may be determined in accordance with the present invention. Changes of molecular weight occurring in the course of an enzymatic processing of DNA or other nucleic acids may therefore be determined by monitoring changes in electrical conductivity of the solution or reaction mixture .
  • electrical conductivity may be conveniently measured as the electrical current flowing through a solution of the nucleic acid material .
  • the present invention is of primary interest for the determination of properties or changes in properties in a single species of nucleic acid.
  • absolute purity of the material is not always necessary and the invention is applicable to nucleic acids containing minor amounts or other materials, including other species of nucleic acid.
  • the concentration of DNA may be determined by measuring the current/conductivity at a known alternating current frequency.
  • the current recorded at any one fixed frequency has been found to be proportional to the DNA concentration.
  • Figure 1 shows how the current/conductivity recorded at 2KHz varies with DNA concentration. This relationship applies not only to a single size of nucleic acid but is true for a range of different sized molecules.
  • a conductivity meter may be readily adapted and calibrated in accordance with the predetermined relationship between current flow and concentration of nucleic acid. In practice it will usually be desirable to calibrate the instrument to deal with homogeneous DNA species but for a range of molecular weights. Thus the sample will normally first be "sized” following which the appropriate nucleic setting for concentration determination will be chosen.
  • the molecular weight of a DNA species is determined by the response of these molecules to varying frequencies applied across the electrodes. Plotting the current/conductivity recorded (Table 1) as percentage of the maximum current /conduct ivity response ( % response) , versus the frequency cf the a.c. signal applied between the two electrodes gives characteristic curves which differ for the molecular weights of the molecules concerned ( Figure 2) .
  • the molecular weight can be determined by firstly calculating the gradient of the response versus frequency curve (over the frequency range 0 - 5xl0 5 Hz) . The gradient value can then be compared with a calibration curve (figure 3) of log molecular weight plotted against gradient. For all DNA molecules tested, the gradient varies with molecular weight such that the larger the gradient value, the lower the molecular weight . The basis of this relationship is presumed to be that the mobility of the DNA molecule changes as the frequency changes,- as the frequency increases, large molecules are less responsive to changes compared with smaller molecules .
  • Methods of DNA quantification and molecular weight determination in accordance with the invention circumvent the problems associated with the known methodologies and offer a number of distinct advantages over those conventional methods.
  • the invention allows rapid and accurate determination of both molecular weight and concentration,- it is more sensitive and accurate, and it requires small sample volumes.
  • a.c. signal of modulating frequency
  • two thin wire platinum electrodes alternate metals such as copper, stainless steel would also be adequate
  • a fixed a.c. signal of between 0 to 10 volts is applied between the two thin wire conductive electrodes.
  • a range of frequencies of this a.c. signal between 0 and 1MHz, is applied across these electrodes and the corresponding conductivity recorded at each frequency in turn as the current passing through the solution.
  • the sample DNA is dissolved in water or TE buffer in a volume of at least 10 ⁇ l .
  • a standard 500 ⁇ l plastic tube can be used.
  • Two platinum thin wire electrodes are placed in the solution and connected to a function generator operating at 1 V a.c.
  • a range of frequencies from 0-1 MHz are passed through the solution and the resulting current measured as mA using an ammeter.
  • the DNA concentration is calculated by comparing the current passing through the solution at a frequency of 2 KHz with a standard curve (relating DNA concentration to current at a fixed frequency) .
  • Alternative frequencies can also be used.
  • Figure 4 shows the mean % response date from table 3 plotted as a graph.
  • the most obvious difference between the DNA solutions is the gradient of the slopes.
  • this gradient is calculated from the conductivity at 2 x 10 and 4 x 10 5 a near linear relationship is observed between gradient and log molecular weight (figure 5) .
  • gradient can be used to calculate molecular weight.
  • PCR is a major molecular biological tool both in academic research and in medical diagnostics, where PCR is used to distinguish individuals who carry specific genetic traits.
  • PCR is a major molecular biological tool both in academic research and in medical diagnostics, where PCR is used to distinguish individuals who carry specific genetic traits.
  • One of the major bottle-necks encountered in medical diagnostics is the analysis of the PCR products. This usually involves a single pure DNA product which needs to be sized accurately. This is normally carried out by laborious gel electrophoresis .
  • Measurements of conductivity have also been applied to PCR.
  • a fixed frequency a.c. signal (0 and 1MHz - 0 to 10 volts) may be passed between two thin wire platinum electrodes as above and the current/conductivity measured through the PCR reaction mix as the reaction proceeds. Measurements may be taken at fixed points during the reaction, or constantly. Conductivity fluctuations during the PCR follow distinct patterns which allow real-time assessment of the reaction. Initially there is a drop in conductivity, probably caused by dissociation and denaturation of the genomic template DNA. Successful reactions show a rapid rise in conductivity over the proceeding cycles which reach a plateau which signifies that the end of the reaction has been reached ( Figure 4) . Unsuccessful reactions show no such increase in conductivity over the proceed- ing cycles. Experimental details and results obtained are described below.
  • This increase in conductivity during the final cycles of the reaction can be attributed to an increase in the yield of the reaction.
  • the decrease in conductivity during the initial cycles may be accounted for by changes in the concentration of small conducting species such as primers, nucleotides and inorganic ions, whose mobilities contribute to a greater extent than large DNA molecules at the high ac frequencies used.
  • concentration of product increases it begins to contribute to the overall conductivity of the solution, thus at high DNA concentrations the conductivity of the reaction mixture increases.
  • the concentration of DNA begins to level off and the conductance of the solution reaches a plateau.
  • a method for the estimation of a property or parameter of a nucleic acid material, or of a process in which a nucleic acid is modified by a chemical or biochemical reaction said property or p.ar.ameter being one to which the electrical conductivity of the nucleic acid material is related, which method comprises measuring the electrical conductivity of the nucleic acid material, or of a reaction mixture containing said material, and estimating from said measurement the property or parameter of the material or process by reference to a predetermined relationship between electrical conductivity .and said property or p.arameter.
  • the parameter estimated may be the concentration of a simple species of nucleic acid in a solution thereof.
  • the property estimted my be the molecule weight of a nucleic acid, estimated from the predetermined relationship between molecule weight and the characteristic curve of electrical current/frequency response of a part thereof. The relationship may be between molecular weight and the area under the ch.aracteristic curve of frequency response.
  • the property estimated is an overall indicator of molecular weight for a range of nucleic acid species.
  • the measurement may be carried out with apparatus which has been precalibrated in accordance with the predetermined relationship, which may be both the molecular weight/conductivity relationship and the concentration/conductivity relationship.
  • said parameter may be the extent to which an enzymatic processing of nucleic acid has proceeded, such as a polymerase chain reaction, a reverse transcription, or a nucleic acid ligation.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention se rapporte à un procédé destiné à évaluer une propriété ou un paramètre d'un matériau nucléotidique ou bien un processus dans lequel un acide nucléique est modifié au terme d'une réaction chimique ou biologique. La propriété ou le paramètre en question doit être un rapport avec la conductivité électrique du matériau nucléotidique. Le procédé consiste à mesurer la conductivité électrique du matériau nucléotidique ou d'un mélange de réaction qui contient ce matériau et, sur la base de ces mesures, à évaluer la propriété ou le paramètre du matériau ou du processus en se référant à un rapport prédéterminé entre la conductivité électrique et la propriété ou le paramètre recherchés.
EP98939776A 1997-08-22 1998-08-21 Evaluation d'un acide nucleique Withdrawn EP0990050A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9717932.9A GB9717932D0 (en) 1997-08-22 1997-08-22 Estimation of nucleic acid
GB9717932 1997-08-22
PCT/GB1998/002516 WO1999010530A1 (fr) 1997-08-22 1998-08-21 Evaluation d'un acide nucleique

Publications (1)

Publication Number Publication Date
EP0990050A1 true EP0990050A1 (fr) 2000-04-05

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP98939776A Withdrawn EP0990050A1 (fr) 1997-08-22 1998-08-21 Evaluation d'un acide nucleique

Country Status (6)

Country Link
EP (1) EP0990050A1 (fr)
JP (1) JP2001514376A (fr)
CN (1) CN1267336A (fr)
CA (1) CA2300868A1 (fr)
GB (1) GB9717932D0 (fr)
WO (1) WO1999010530A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9928232D0 (en) * 1999-12-01 2000-01-26 Skelton Stephen Detection system
GB0002859D0 (en) * 2000-02-08 2000-03-29 Molecular Sensing Plc Process for characterising nucleic acids in solution
WO2001081619A2 (fr) * 2000-04-22 2001-11-01 Borros Arneth Pcr avec mesure de conductivite
KR100858080B1 (ko) 2002-11-12 2008-09-10 삼성전자주식회사 전기적 신호를 측정하는 ｐｃｒ 증폭 산물을 검출하는 방법
GB0905325D0 (en) 2009-03-30 2009-05-13 Selex Sensors & Airborne Sys Detection system
CN102141533B (zh) * 2010-11-26 2013-01-02 中国水产科学研究院黄海水产研究所 环介岛等温基因扩增结果分析方法
JP5712105B2 (ja) * 2011-10-14 2015-05-07 株式会社ジャパンディスプレイ 液晶表示装置
US9019439B2 (en) 2011-10-14 2015-04-28 Japan Display Inc. Liquid crystal display device

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60260838A (ja) * 1984-06-08 1985-12-24 Koji Ando オイルの品質測定装置
DE3513168A1 (de) * 1985-04-12 1986-10-16 Thomas 8000 München Dandekar Biosensor bestehend aus einem halbleiter auf silizium oder kohlenstoffbasis (elektronischer teil) und nukleinbasen (od. anderen biol. monomeren)
FR2598227B1 (fr) * 1986-04-30 1989-07-28 Bio Merieux Procede de detection et/ou d'identification d'une substance biologique dans un milieu liquide a l'aide de mesures electriques, et dispositif destine a la mise en oeuvre de ce procede
GB2248301A (en) * 1990-09-12 1992-04-01 Ici Plc Apparatus and method for the detection of changes in the composition of a material
GB9215733D0 (en) * 1992-07-24 1992-09-09 British Tech Group Method of and apparatus for determining a property of a sample
JPH08505476A (ja) * 1993-03-05 1996-06-11 ユニヴァーシティー オブ ウーロンゴング 電気的に活性なポリマ電極を用いたパルス式電気化学的検出方法
GB2289338A (en) * 1994-04-12 1995-11-15 Unvala Ltd Alternating current potential drop measurement
CA2238003C (fr) * 1995-12-01 2005-02-22 Innogenetics N.V. Systeme de detection par mesure de l'impedance et procede pour le fabriquer
GB9604292D0 (en) * 1996-02-29 1996-05-01 Hybaid Ltd Estimation of a nucleic acid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9910530A1 *

Also Published As

Publication number Publication date
WO1999010530A1 (fr) 1999-03-04
CN1267336A (zh) 2000-09-20
CA2300868A1 (fr) 1999-03-04
JP2001514376A (ja) 2001-09-11
GB9717932D0 (en) 1997-10-29

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