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WO1998002573A1 - Denaturation de l'acide nucleique a double brin - Google Patents

Denaturation de l'acide nucleique a double brin Download PDF

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Publication number
WO1998002573A1
WO1998002573A1 PCT/GB1997/001865 GB9701865W WO9802573A1 WO 1998002573 A1 WO1998002573 A1 WO 1998002573A1 GB 9701865 W GB9701865 W GB 9701865W WO 9802573 A1 WO9802573 A1 WO 9802573A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
voltage
denaturation
electrode
electrodes
Prior art date
Application number
PCT/GB1997/001865
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English (en)
Inventor
Duncan Ross Purvis
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Scientific Generics Limited
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
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Publication of WO1998002573A1 publication Critical patent/WO1998002573A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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

  • Th s invention relates to processes for the treatment of nucleic acid material m order to effect a complete or partial change from double-stranded form to single-stranded form and to processes of amplifying or detecting nucleic acids involving such denaturation processes.
  • Double-stranded DNA (deoxyr bonucleic acid) and DNA/RNA (ribonucleic acid) and RNA/RNA complexes in the familiar double helical configuration are stable molecules that, in vitro, require aggressive conditions to separate the complementary strands of the nucleic acid.
  • Known methods that are commonly employed for strand separation require the use of high temperatures of at least 60°C and often 100°C or use an alkaline pH of 11 or higher.
  • Other methods include the use of helicase enzymes such as Rep protein of E.coli that can catalyse the unwinding of the DNA in an unknown way, or binding proteins such as 32 -protein of E.coli phage T4 that act to stabilise the single-stranded form of DNA.
  • the denatured single-stranded DNA produced by the known processes of heat cr alkali treatment is used commonly for hybridisation studies or is suDjected to amplification cycles.
  • Such separation is a prerequisite of a number of protocols involving the in vitro manipulation of nucleic acids, one example of which is a reaction that produces multiple copies of target sequences of DNA and which employs a heat-stable polymerase enzyme (US Patent No. 4683202, K.B. ullis et al) .
  • This development known as the polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • strand separation is normally effected by heating the sample to approximately 95 °C.
  • the removal of the need to heat the sample would provide a number of benefits. For example, it allows the design of compact and readily controllable apparatus, and the use of higher fidelity mesophyllic enzymes.
  • WO 92/04470 discloses a process whereby nucleic acid strands are separated by the application of an electric field. The advantages of the electrical method are discussed in greater detail, along with the method's application in ampli- fication reactions such as PCR and ligase chain reaction. Forms of electrochemical cells for carrying out the reaction are described and also the use of "promoter" compounds that enhance the efficiency of denaturation.
  • a PCR reaction is conducted in which there are repeated denaturation operations conducted using the electrochemical cell described with intervening amplification stages.
  • the denaturation stages are each conducted for a period of five minutes or longer and the total time for the PCR reaction is therefore very extended.
  • the conditions under which the PCR reaction was conducted in WO92/00470 differ from those of the conventional PCR process in that it was not found possible to use a conventional PCR buffer system. In order to obtain denaturation, it was necessary to conduct the process at a much lower ionic strength than would be consistent with such a buffer system. Excluding the promoter methyl viologen, the process was basically conducted in distilled water.
  • W095/25177 contains examples in which complete denaturation of DNA is achieved within 1 to 2 minutes in comparison with denaturation times of at least 25 minutes using the electrode set up of WO92/04470.
  • the positively charged viologen molecules interact between the negatively charged DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the DNA to the electrode surface where the electrical field is at its stronger. Accordingly, we expressed a preference in WO92/04470 to employ as promoters compounds having spaced positively charged centres, e.g. bipolar positively charged compounds. Preferably, the spacing between the positively charged centres was to be similar to that in viologens .
  • W093/15224 was in turn based on the discovery that multivalent inorganic cations, preferably Mg 2+ , can also act as promoters in such a system with approximately the same efficacy as methyl viologen.
  • lithium ions can also promote denaturation.
  • the present invention provides a process for denaturing double-stranded nucleic acid which comprises applying a voltage to a solution containing said nucleic acid with an electrode under conditions such as a convert a substantial proportion of said nucleic acid to a wholly or partially single-stranded form wherein the solution contains an effective concentration of lithium ions acting as a promoter which assists said denaturation.
  • the concentration of said promoter cation is preferably from 1 mM to 50 mM, more preferably from 5 mM to 20 ttiM, e.g. about 10 mM.
  • the nucleic acid does not have to be dissolved in the solution but may be immobilised to a solid phase immersed in the solution,
  • the single-stranded nucleic acid produced is free from the electrode, e.g. dissolved in the solution.
  • the nucleic acid may be immobilised on the electrode in double or single-stranded form prior to the application of the electric potential, e.g. attached by the end or a small portion intermediate the ends of the nucleic acid chain, so as to leave substantial segments of the nucleic acid molecules freely pendant from the electrode surface before and after denaturation.
  • a reference electrode may be contacted with said solution and a voltage may be applied between said electrode and said counter-electrode so as to achieve a desired controlled voltage between said electrode and said reference electrode.
  • the electrodes may be connected by a potentiostat circuit as is known in the electrochemical art.
  • a potential of from -0.5 to -1.5 V is applied to said working electrode with respect to said reference electrode, more preferably from -0.8 to -1.1 V, e.g. about - 1.0 V.
  • Working electrode voltages relative to reference electrodes are given throughout as if measured or as actually measured relative to a calomel reference electrode (BDH No. 309.1030.02) .
  • the process may be conducted using a three electrode system of the kind described in WO92/04470 but generally it is preferred that the volume of solution employed according to this invention is small e.g. 1 ml or less, preferably very small e.g. 100 ⁇ l or less, e.g. about 25 ⁇ l to 40 ⁇ l.
  • a voltage will be applied between two electrodes and will be measured directly.
  • Voltages given herein for two electrode systems are given in this way and not with reference to a calomal electrode. It is preferred to apply a voltage difference of from 0.5 to 3 volts between the electrodes. Voltage differences above 3 volts seem to inhibit denaturation or promote degradation although the mechanism involved here is presently unknown. Preferably, the process is conducted at a voltage of 1.5 to 2.5 volts measured as a voltage difference between the electrodes .
  • a constant current regime it will generally be preferable to use a current of from 80 to 160 ⁇ A, e.g. about 100 to 125 ⁇ A.
  • a promoter compound such as methyl viologen to produce more rapid denaturation.
  • Other promoters are described in W093/15224, i.e. multivalent cations such as magnesium.
  • multivalent cations which are effective and which can be used include lanthanum (La 3 + ) .
  • the cations used as the promoters may include inorganic cations complexed with inorganic or organic ligands, e.g. Pt(NH 3 ) 6 4+ and Cr(NH 3 ) 6 2+ .
  • Such a promoter may be any inorganic or organic molecule which increases the rate of extent of denaturation of the double helix.
  • the promoter may be immobilised to or included in material from which the electrode is constructed.
  • the additional promoter may be a water-soluble compound of the bipyridyl series, especially a viologen such as methyl - viologen or a salt thereof. Whilst the mechanism of operation of such promoters is presently not known with certainty, it ⁇ s believed that the positively charged viologen molecules interact between the negatively charged nucleic acid such as DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the DNA to the electrode surface where the electrical field is at its strongest. Accordingly, we prefer to employ as promoters compounds having spaced positively charged centres, e.g. bipolar positively charged compounds. Preferably the spacing between the positively charged centres is similar to that m viologens. Other suitable viologens include ethyl - viologen, isopropyl -viologen and benzyl -viologen.
  • the ionic strength of said solution is preferably no more than 250 mM, more preferably no more than 100 mM. As it has been found that the rate of denaturation increases as the ionic strength is decreased, the said ionic strength is still more preferably no more than 50 mM, e.g. no more than 25 mM or even no more than 5 mM. Generally, the lower the ionic strength, the more rapid is the denaturation. However, in calculating ionic strength for these purposes it may be appro- priate to ignore the contribution to ionic strength of any component which acts as a promoter as described above .
  • the electrode may be a so called "modified electrode” m which the denaturing is promoted by a compound coated on to, or adsorbed on to, or incorporated into the structure of the electrode which is otherwise of an inert but conducting material .
  • electrochemical cell for use m this invention uses a carbon rod electrode dipping into a carbon block containing a well.
  • working, counter and optionally reference electrodes may be formed on a single surface, e.g. a flat surface by any printing method such as thick film screen printing, ink j-et printing, or by using a photo-resist followed by etching.
  • the counter and reference electrodes can be combined on the flat surface leading to a two electrode configuration.
  • the electrodes may be formed on the inside surface of a well which is adapted to hold liquid, such a well could be the well known 96 well or Microtitre plate, it may also be a test tube or other vessel. Electrode arrays in Microtitre plates or other moulded or thermofor ed plastic materials may be provided for multiple nucleic acid denaturation experiments.
  • the strand separation may be carried out in an aqueous medium or in a mixture of water with an organic solvent such as dimethylformamide.
  • the use of polar solvents other than water or non-polar solvents is also accepted but is not preferred.
  • the process may be carried out at ambient temperatures or if desired temperatures up to adjacent the pre- melting temperature of the nucleic acid.
  • the process may be carried out at pH's of from 3 to 10 conveniently about 7. Generally, more rapid denaturation is obtained at lower pH. For some purposes therefore a pH somewhat below neutral, e.g. about pH 5.5 may be preferred.
  • the nucleic acid may be dissolved in an aqueous solution containing a buffer whose nature and ionic strength are such as not to interfere with the strand separation process.
  • the denaturing process according to the invention may be incorporated as a step in a number of more complex processes, e.g. procedures involving the analysis and/or the amplifi- cation of nucleic acid. Some examples of such applications are described below.
  • the invention includes a process for detecting the presence or absence of a predetermined nucleic acid sequence in a sample which comprises: denaturing a sample double- stranded nucleic acid by means of an electrode; hybridising the denatured nucleic acid with an oligonucleotide probe for the sequence; and determining whether the said hybridisation has occurred, wherein during denaturing the solution contains an effective concentration of lithium ion acting as a promoter which assists said denaturation.
  • the invented process has application in DNA and RNA hybridisation where a specific gene sequence is to be indenti- fied e.g. specific to a particular organism or specific to a particular hereditary disease of which sickle cell anaemia is an example.
  • a sample of DNA preferably of purified DNA, means for which are known, which is in native double-stranded form. It is then necessary to convert the double-stranded DNA to single- stranded form before a hybridisation step with a labelled nucleotide probe which has a complementary sequence to the DNA sample can take place.
  • the denaturation process of the invention can be used for this purpose in a preferred manner by carrying out the following steps: denaturing a sample of DNA by applying a voltage by means of an electrode to the sample DNA with a said promoter in solution;
  • a typical DNA probe assay it is customary to immobilise the sample DNA to a membrane surface which may be composed of neutral or charged nylon or nitrocellulose. The immobilisation is achieved by charge interactions or by baking the membrane containing DNA in an oven.
  • the sample DNA can be heated to high temperature to ensure conversion to single- stranded form before binding to the membrane or it can be treated with alkali once on the membrane to ensure conversion to the single-stranded form.
  • the disadvantages of the present methods are : heating to high temperatures to create single-stranded
  • DNA can cause damage to the sample DNA itself, - the use of alkali requires an additional step of neutralisation before hybridisation with the labelled probe can take place.
  • One improved method for carrying out DNA probe hybridisation assays is the so called "sandwich" technique where a specific oligonucleotide is immobilised on a surface.
  • the surface having the specific oligonucleotide thereon is then hybridised with a solution containing the target DNA in a single-stranded form, after which a second labelled oligonucleotide is then added which also hybridises to the target DNA.
  • the surface is then washed to remove unbound labelled oligonucleotide, after which any label which has become bound to target DNA on the surface can be detected later.
  • This procedure can be simplified by using the denaturing process of the invention to denature the double-stranded DNA into the required single-stranded DNA.
  • the working electrode, counter electrode and optionally a reference electrode and/or the promoter can be incorporated into a test tube or a well in which the DNA probe assay is to be carried out.
  • the DNA sample, promoter if not already present and oligonucleotide probes can then be added and the voltage applied to denature the DNA.
  • the resulting single-stranded DNA is hybridised with the specific oligonucleotide immobilised on the surface after which the remaining stages of a sandwich assay are carried out . All the above steps can take place without a need for high temperatures or addition of alkali reagents as in the conventional process.
  • the electrochemical denaturation of DNA can be used in the amplification of nucleic acids, e.g. in a polymerase chain reaction or ligase chain reaction amplification procedure.
  • the present invention provides a process for replicating a nucleic acid which comprises: separating the strands of a sample double-stranded nucleic acid in solution under the influence of a lithium ion promoter and an electrical voltage applied to the solution from an electrode; hybridising the separated strands of the nucleic acid with at least one oligonucleotide primer that hybridises with at least one of the strands of the denatured nucleic acid; synthesising an extension product of and or each primer which is sufficiently complementary to the respective strand of the nucleic acid to hybridise therewith; and separating the or each extension product from the nucleic acid strand with which it is hybridised to obtain the extension product.
  • the said amplification process further comprises repeating the procedure defined above cyclicly, e.g. for more than 10 cycles, e.g. up to 20 or 30 cycles.
  • the hybridisation step is preferably carried out using two primers which are complementary to different strands of the nucleic acid.
  • the denaturation to obtain the extension products as well as the original denaturing of the target nucleic acid is preferably carried out by applying to the solution of the nucleic acid a voltage from an electrode, the solution containing lithium ion as promoter as described herein.
  • the process may be a standard or classical PCR process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process comprises : (a) treating the strands with two oligonucleotide primers, for each different specific sequence being applied, under conditions such that for each different sequence being amplified an extension product of each primer is synthesised which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to different strands of each specific sequence such that the extension product synthesised from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension produce of the other primer;
  • step (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesised using each of the single strands produced in step (b) as a template.
  • the process may be any variant of the classical or standard PCR process, e.g. the so-called “inverted” or “inverse” PCR process or the "anchored” PCR process .
  • the invention therefore includes an amplification process as described above in which a primer is hybridised to a circular nucleic acid and is extended to form a duplex which is denatured by the denaturing process of the invention, the amplification process optionally being repeated through one or more additional cycles.
  • the invention includes a process for replicating a target sequence of nucleic acid comprising hybridisation, extension and denaturation of nucleic acid (e.g. cycles of hybridising and denaturing) wherein said denaturation is produced by operating on a solution containing said nucleic acid with an electrode in the presence of lithium ion as promoter.
  • the process of the invention is applicable to the ligase chain reaction. Accordingly, the invention includes a process for amplifying a target nucleic acid comprising the steps of:
  • the first and second of said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes; ii) the first probe is a single strand capable of hybridising to a first segment of a primary strand of the target nucleic acid; iii) the second probe is a single strand capable of hybridising to a second segment of said primary strand of the target nucleic acid; iv) the 5' end of the first segment of said primary strand of the target is positioned relative to the
  • the third probe is capable of hybridising to the first probe; and vi) the fourth probe is capable of hybridising to the second probe ; and (c) repeatedly or continuously: i) hybridising said probes with nucleic acid in said sample; ii) ligating hybridised probes to form reorganised fused probe sequences ; and
  • the denaturation of the DNA to allow subsequent hybridisation with the primers can be carried out by the application of an appropriate potential to the electrode.
  • the process may be carried out stepwise involving successive cycles of denaturation or renaturation as in the existing thermal methods of PCR and LCR, but it is also possible for it to be carried out continuously since the process of chain extension or ligation by the enzyme and subsequent strand separation by the electro- chemical process can continue in the same reaction as nucleic acid molecules in single-stranded form will be free to hybridise with primers once they leave the denaturing influence of the electrode.
  • the electrochemical DNA amplification technique can be used analytically to detect and analyse a very small sample of DNA e.g. a single copy gene in an animal cell or a single cell of a bacterium.
  • the time required for denaturation to occur may be extremely short, e.g. less than 0.01 second up to 0.1 second.
  • a process of repeated denaturation of double-stranded nucleic acid may be performed, in which said voltage is applied as a repeating pulse having a duration of up to 2 minutes, e.g. up to one minute or much less . Between said pulses the voltages may be turned off and/or reversed for a period similar to or equal to the period for which the voltage is applied, e.g. the voltage may be applied as pulses at a frequency of from 0.01 to 100 Hz.
  • a single denaturation may be performed using a single pulse cycle.
  • the voltage may be applied such that there are, in other any order, periods of application of voltage with a first polarity, periods of application of voltage with the opposite polarity to said first polarity and periods of substantially reduced applied voltage.
  • the cycles may be from 0.01 seconds to 5 minutes or more in length, e.g. from 1 second to 5 minutes in length.
  • the periods during which said voltage is applied with a first polarity and said periods during which said voltage is applied with a second polarity are each independent of from 0.5 seconds to 1 minute.
  • the periods during which said voltage is substantially reduced are each individually from 0.5 seconds to 3 minutes.
  • the invention includes a kit for use in a process of detecting the presence or absence of a predetermined nucleic acid sequence in a sample which kit comprises, an electrode, a counter electrode and optionally a reference electrode, an oligonucleotide probe for said sequence and a source of an lithium ion for use as a promoter in obtaining nucleic acid strand separation at said electrode.
  • the probe may be labelled in any of the ways discussed above.
  • the invention also includes a kit for use in a process of nucleic acid amplification comprising an electrode, a counter electrode and optionally a reference electrode, and a source of lithium ion for use as a promoter in obtaining nucleic acid strand separation at said electrode and at least one primer for use iri a PCR procedure, or at least one primer for use in an LCR procedure, and/or a polymerase or a ligase, and/or nucleotide suitable for use in a PCR process.
  • a kit for use in a process of nucleic acid amplification comprising an electrode, a counter electrode and optionally a reference electrode, and a source of lithium ion for use as a promoter in obtaining nucleic acid strand separation at said electrode and at least one primer for use iri a PCR procedure, or at least one primer for use in an LCR procedure, and/or a polymerase or a ligase, and/or nucleotide suitable for use in
  • kits includes a cell containing the electrodes.
  • the kits include a suitable buffer for use in the detection or amplification procedure.
  • Figure 1 is a diagram of an electrochemical cell used for denaturation of DNA.
  • Figure 2 is a gel produced in the Example.
  • the cell shown in Figure 1 comprises a graphite block 10 fabricated from 6B pencil graphite containing a 3 mm diameter well 12, which is 15 mm deep, having at ts upper end an enlarged diameter mouth portion 13.
  • a probe electrode 14 is formed from an 2B to 2H (e.g. HB) pencil graphite and is 2 mm in diameter. It is supported in the wflll 12 by a sleeve 15 of PTFE in a base in which it is tightly received. A pair of O- rings 19 received in grooves in the sleeve 15 provide a snug fit for the sleeve 15 in the mouth portion 13 of the block 10.
  • the precise height of the probe electrode m the well can be set by protruding the desired extent from the lower end of the sleeve 15.
  • the end of the probe electrode is flat and not tapered.
  • a block containing the wells has a liftable l d with electrodes depending therefrom into the wells.
  • Each well may have a pair of electrodes on the lid in various possible electrode conformations such as parallel rods, parallel plates, optionally of mesh, or coaxial hollow cylinders, again optionally of mesh.
  • single electrodes may be provided on the lid for each well and the block containing the wells may be conductive and may serve as a common second electrode.
  • the block of wells may also contain respective electrodes of each well.
  • the lid may comprise a flat plate portion bearing the electrode or electrodes for each well and a separate backing member bearing electrical connections and circuitry which connects up the electrodes when the two parts are assembled.
  • a single electrical supply to the unit may be split by said circuitry and supplied in a controlled manner to the electrodes such that each electrode is controlled in voltage.
  • the plate portion carrying the electrode array may thereby be replaceable without replacement of the control circuitry and may be disposable.
  • the plate portion and the backing member may be aligned with one another on assembly by locating pins and apertures and may similarly be aligned with the block containing the wells, which also may be disposable.
  • the cell shown in Figure 1 is used in the following example .
  • EXAMPLE 1 To the working chamber of the cell shown in Figure 1 was added 50 ⁇ l and 10 ⁇ g/ml Calf Thymus DNA in 10 mM Tris HClat pH 7.0 together with the amount of promoter or control salt shown in Table 1 below. The contents of the cell were subjected to a voltage of 2.4 V for the period shown in the table.

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Abstract

La dénaturation électrochimique de l'ADN et son passage à une forme à simple brin, provoqués par l'application d'un courant électrique variable, sont accélérés par la présence d'ions lithium.
PCT/GB1997/001865 1996-07-11 1997-07-10 Denaturation de l'acide nucleique a double brin WO1998002573A1 (fr)

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GBGB9614544.6A GB9614544D0 (en) 1996-07-11 1996-07-11 Denaturation of double-stranded nucleic acid

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044100A1 (fr) * 1997-04-02 1998-10-08 Scientific Generics Limited Dissociation de molecules reagissant mutuellement
US7494791B2 (en) 2004-05-13 2009-02-24 Nanobiosym, Inc. Nano-PCR: methods and devices for nucleic acid amplification and detection
US9862984B2 (en) 2006-04-21 2018-01-09 Nanobiosym, Inc. Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents
US10280392B2 (en) 2013-03-14 2019-05-07 The Regents Of The University Of California Methods and devices for non-thermal polymerase chain reaction
US10933417B2 (en) 2013-03-15 2021-03-02 Nanobiosym, Inc. Systems and methods for mobile device analysis of nucleic acids and proteins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004470A1 (fr) * 1990-09-12 1992-03-19 Scientific Generics Limited Denaturation electrochimique d'acide nucleique bicatenaire
WO1993015224A1 (fr) * 1992-01-23 1993-08-05 Scientific Generics Limited Traitement electrochimique de solutions contenant de l'acide nucleique
WO1995025177A1 (fr) * 1994-03-15 1995-09-21 Scientific Generics Limited Dénaturation électro-chimique de l'acide nucléique double brins
WO1997008293A1 (fr) * 1995-08-25 1997-03-06 Scientific Generics Limited Liberation de matiere intracellulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004470A1 (fr) * 1990-09-12 1992-03-19 Scientific Generics Limited Denaturation electrochimique d'acide nucleique bicatenaire
WO1993015224A1 (fr) * 1992-01-23 1993-08-05 Scientific Generics Limited Traitement electrochimique de solutions contenant de l'acide nucleique
WO1995025177A1 (fr) * 1994-03-15 1995-09-21 Scientific Generics Limited Dénaturation électro-chimique de l'acide nucléique double brins
WO1997008293A1 (fr) * 1995-08-25 1997-03-06 Scientific Generics Limited Liberation de matiere intracellulaire

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Title
C.J. STANLEY ET AL.: "Amperometric enzyme-amplified immunoassays", J. IMMUNOL. METHODS, vol. 112, no. 2, 1988, ELSEVIER, AMSTERDAM NL, pages 153 - 161, XP002043865 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044100A1 (fr) * 1997-04-02 1998-10-08 Scientific Generics Limited Dissociation de molecules reagissant mutuellement
US6333157B1 (en) * 1997-04-02 2001-12-25 Affymetrix, Inc. Disassociation of interacting molecules
US7494791B2 (en) 2004-05-13 2009-02-24 Nanobiosym, Inc. Nano-PCR: methods and devices for nucleic acid amplification and detection
US8632973B2 (en) 2004-05-13 2014-01-21 Nanobiosym, Inc. Nano-PCR: methods and devices for nucleic acid amplification and detection
US9862984B2 (en) 2006-04-21 2018-01-09 Nanobiosym, Inc. Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents
US11807892B2 (en) 2006-04-21 2023-11-07 Nanobiosym, Inc. Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents
US12435351B2 (en) 2006-04-21 2025-10-07 Nanobiosym, Inc. Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents
US10280392B2 (en) 2013-03-14 2019-05-07 The Regents Of The University Of California Methods and devices for non-thermal polymerase chain reaction
US10933417B2 (en) 2013-03-15 2021-03-02 Nanobiosym, Inc. Systems and methods for mobile device analysis of nucleic acids and proteins

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