WO1999010530A1 - Estimation of nucleic acid - Google Patents
Estimation of nucleic acid Download PDFInfo
- Publication number
- WO1999010530A1 WO1999010530A1 PCT/GB1998/002516 GB9802516W WO9910530A1 WO 1999010530 A1 WO1999010530 A1 WO 1999010530A1 GB 9802516 W GB9802516 W GB 9802516W WO 9910530 A1 WO9910530 A1 WO 9910530A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- conductivity
- parameter
- molecular weight
- property
- 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
- C12Q1/6813—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.
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- 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
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000507837A JP2001514376A (en) | 1997-08-22 | 1998-08-21 | Nucleic acid estimation |
CA002300868A CA2300868A1 (en) | 1997-08-22 | 1998-08-21 | Estimation of nucleic acid |
EP98939776A EP0990050A1 (en) | 1997-08-22 | 1998-08-21 | Estimation of nucleic acid |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9717932.9 | 1997-08-22 | ||
GBGB9717932.9A GB9717932D0 (en) | 1997-08-22 | 1997-08-22 | Estimation of nucleic acid |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999010530A1 true WO1999010530A1 (en) | 1999-03-04 |
Family
ID=10817954
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1998/002516 WO1999010530A1 (en) | 1997-08-22 | 1998-08-21 | Estimation of nucleic acid |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0990050A1 (en) |
JP (1) | JP2001514376A (en) |
CN (1) | CN1267336A (en) |
CA (1) | CA2300868A1 (en) |
GB (1) | GB9717932D0 (en) |
WO (1) | WO1999010530A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001059154A2 (en) * | 2000-02-08 | 2001-08-16 | Molecular Sensing Plc | Process for characterising nucleic acids in solution |
WO2001081619A2 (en) * | 2000-04-22 | 2001-11-01 | Borros Arneth | Conductivity pcr |
WO2001040511A3 (en) * | 1999-12-01 | 2002-05-10 | Secr Defence | Detection system |
EP1420070A1 (en) * | 2002-11-12 | 2004-05-19 | Samsung Electronics Co., Ltd. | Method for detecting PCR products from electrical signal generation |
WO2010112495A1 (en) | 2009-03-30 | 2010-10-07 | Selex Sensors And Airborne Systems Limited | Detection system comprising a light guide for directing radiation to a reaction chamber |
JP2013088515A (en) * | 2011-10-14 | 2013-05-13 | Japan Display Central Co Ltd | Liquid crystal display device |
US9019439B2 (en) | 2011-10-14 | 2015-04-28 | Japan Display Inc. | Liquid crystal display device |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102141533B (en) * | 2010-11-26 | 2013-01-02 | 中国水产科学研究院黄海水产研究所 | Method for analyzing result of loop-mediated isothermal amplification |
Citations (9)
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JPS60260838A (en) * | 1984-06-08 | 1985-12-24 | Koji Ando | Instrument for measuring oil quality |
DE3513168A1 (en) * | 1985-04-12 | 1986-10-16 | Thomas 8000 München Dandekar | BIOSENSOR CONSISTING OF A SEMICONDUCTOR BASED ON SILICON OR CARBON-BASED (ELECTRONIC PART) AND NUCLEIN BASE (OR. OTHER BIOL. MONOMERS) |
EP0244326A2 (en) * | 1986-04-30 | 1987-11-04 | BIO MERIEUX Société anonyme dite: | Method for detecting and/or identifying a biological substance in a liquid medium with the aid of electrical measurements, and apparatus for carrying out this method |
GB2248301A (en) * | 1990-09-12 | 1992-04-01 | Ici Plc | Apparatus and method for the detection of changes in the composition of a material |
WO1994002846A1 (en) * | 1992-07-24 | 1994-02-03 | British Technology Group Ltd. | Method of and apparatus for determining a property of a sample |
US5403451A (en) * | 1993-03-05 | 1995-04-04 | Riviello; John M. | Method and apparatus for pulsed electrochemical detection using polymer electroactive electrodes |
WO1995027896A1 (en) * | 1994-04-12 | 1995-10-19 | Unvala Limited | Measurement |
WO1997021094A1 (en) * | 1995-12-01 | 1997-06-12 | Innogenetics N.V. | Impedimetric detection system and method of production thereof |
WO1997032039A1 (en) * | 1996-02-29 | 1997-09-04 | Molecular Sensors Limited | Method for quantitative determination of a nucleic acid in solution by measurement of the electrical conductivity and apparatus therefor |
-
1997
- 1997-08-22 GB GBGB9717932.9A patent/GB9717932D0/en active Pending
-
1998
- 1998-08-21 WO PCT/GB1998/002516 patent/WO1999010530A1/en not_active Application Discontinuation
- 1998-08-21 JP JP2000507837A patent/JP2001514376A/en active Pending
- 1998-08-21 CA CA002300868A patent/CA2300868A1/en not_active Abandoned
- 1998-08-21 CN CN98808164A patent/CN1267336A/en active Pending
- 1998-08-21 EP EP98939776A patent/EP0990050A1/en not_active Withdrawn
Patent Citations (9)
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JPS60260838A (en) * | 1984-06-08 | 1985-12-24 | Koji Ando | Instrument for measuring oil quality |
DE3513168A1 (en) * | 1985-04-12 | 1986-10-16 | Thomas 8000 München Dandekar | BIOSENSOR CONSISTING OF A SEMICONDUCTOR BASED ON SILICON OR CARBON-BASED (ELECTRONIC PART) AND NUCLEIN BASE (OR. OTHER BIOL. MONOMERS) |
EP0244326A2 (en) * | 1986-04-30 | 1987-11-04 | BIO MERIEUX Société anonyme dite: | Method for detecting and/or identifying a biological substance in a liquid medium with the aid of electrical measurements, and apparatus for carrying out this method |
GB2248301A (en) * | 1990-09-12 | 1992-04-01 | Ici Plc | Apparatus and method for the detection of changes in the composition of a material |
WO1994002846A1 (en) * | 1992-07-24 | 1994-02-03 | British Technology Group Ltd. | Method of and apparatus for determining a property of a sample |
US5403451A (en) * | 1993-03-05 | 1995-04-04 | Riviello; John M. | Method and apparatus for pulsed electrochemical detection using polymer electroactive electrodes |
WO1995027896A1 (en) * | 1994-04-12 | 1995-10-19 | Unvala Limited | Measurement |
WO1997021094A1 (en) * | 1995-12-01 | 1997-06-12 | Innogenetics N.V. | Impedimetric detection system and method of production thereof |
WO1997032039A1 (en) * | 1996-02-29 | 1997-09-04 | Molecular Sensors Limited | Method for quantitative determination of a nucleic acid in solution by measurement of the electrical conductivity and apparatus therefor |
Non-Patent Citations (5)
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DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1981, KUZNETSOV ET AL.: "THE EQUIVALENT CONDUCTIVITY OF AQUEOUS SALT-FREE SOLUTIONS OF DNA INFLUENCE OF UNIVALENT COUNTERIONS", XP002088533 * |
GOUREAU Y ET AL: "EVALUATION OF PLASMA SODIUM CONCENTRATION DURING HEMODIALYSIS BY COMPUTERIZATION OF DIALYSATE CONDUCTIVITY", ASAIO TRANSACTIONS, vol. 36, no. 3, 1 July 1990 (1990-07-01), pages 444 - 447, XP000204537 * |
LAWTON B A ET AL: "DETERMINING THE FAT CONTENT OF MILK AND CREAM USING AC CONDUCTIVITY MEASUREMENTS", MEASUREMENT SCIENCE AND TECHNOLOGY, vol. 4, no. 1, 1 January 1993 (1993-01-01), pages 38 - 41, XP000328757 * |
PATENT ABSTRACTS OF JAPAN vol. 010, no. 140 (P - 458) 23 May 1986 (1986-05-23) * |
WATTS H J ET AL: "REAL-TIME DETECTION AND QUANTIFICATION OF DNA HYDRIDAZATION BY AN OPTICAL BIOSENSOR", ANALYTICAL CHEMISTRY, vol. 67, no. 23, 1 December 1995 (1995-12-01), pages 4283 - 4289, XP000540115 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001040511A3 (en) * | 1999-12-01 | 2002-05-10 | Secr Defence | Detection system |
WO2001059154A2 (en) * | 2000-02-08 | 2001-08-16 | Molecular Sensing Plc | Process for characterising nucleic acids in solution |
WO2001059154A3 (en) * | 2000-02-08 | 2002-03-21 | Molecular Sensing Plc | Process for characterising nucleic acids in solution |
WO2001081619A2 (en) * | 2000-04-22 | 2001-11-01 | Borros Arneth | Conductivity pcr |
WO2001081619A3 (en) * | 2000-04-22 | 2002-05-16 | Borros Arneth | Conductivity pcr |
EP1420070A1 (en) * | 2002-11-12 | 2004-05-19 | Samsung Electronics Co., Ltd. | Method for detecting PCR products from electrical signal generation |
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 |
WO2010112495A1 (en) | 2009-03-30 | 2010-10-07 | Selex Sensors And Airborne Systems Limited | Detection system comprising a light guide for directing radiation to a reaction chamber |
JP2013088515A (en) * | 2011-10-14 | 2013-05-13 | Japan Display Central Co Ltd | Liquid crystal display device |
US9019439B2 (en) | 2011-10-14 | 2015-04-28 | Japan Display Inc. | Liquid crystal display device |
Also Published As
Publication number | Publication date |
---|---|
JP2001514376A (en) | 2001-09-11 |
CA2300868A1 (en) | 1999-03-04 |
GB9717932D0 (en) | 1997-10-29 |
CN1267336A (en) | 2000-09-20 |
EP0990050A1 (en) | 2000-04-05 |
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