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WO1986005815A1 - Sequences d'acide nucleique liees a des materiaux sensibles a des champs magnetiques, et methodes d'analyse et appareils utilisant lesdites sequences liees - Google Patents

Sequences d'acide nucleique liees a des materiaux sensibles a des champs magnetiques, et methodes d'analyse et appareils utilisant lesdites sequences liees Download PDF

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Publication number
WO1986005815A1
WO1986005815A1 PCT/GB1986/000174 GB8600174W WO8605815A1 WO 1986005815 A1 WO1986005815 A1 WO 1986005815A1 GB 8600174 W GB8600174 W GB 8600174W WO 8605815 A1 WO8605815 A1 WO 8605815A1
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magnetic
nucleic acid
mixture
enzyme
particles
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PCT/GB1986/000174
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English (en)
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Hugh Alan Oliver Hill
Mark Jonathan Gear
Stephen Charles Williams
Monika Joanna Green
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Genetics International Inc.
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Publication of WO1986005815A1 publication Critical patent/WO1986005815A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • the present invention is concerned with nucleic acid sequences, assays foe nucleic acids and apparatus for such assay.
  • the assay procedures of the present invention extend to both to detection of the presence of nucleic acids and to the identification of specific nucleic acid molecules, or classes of molecules, by identifying sequences therein, both procedures being needed in many areas of biochemical research and commerce.
  • RNA probes Hybridisation between two single-stranded DNA (or RNA) molecules, which have complementary (or partially complementary) sequences has been one approach to the problem of nucleic acid assay.
  • Molecules for use in such assays are capable of binding to a DNA (or RNA) sequence defined to a particular level of sequence homology, and are known as "DNA probes"(or "RNA probes")
  • RNA probe techniques all have the characteristic that the DNA (RNA) polymer formed by hydridisation between the probe and target sequence is not readily detectable by its inherent biochemical activity. It is therefore necessary to mark the polymer formed with some signal-producing chemical or biochemical species.
  • biotin (Vitamin H) is chemically linked to one or more nucleotides,i.e residues which comprise the onomeric units of a DNA polymer.
  • nucleotides i.e residues which comprise the onomeric units of a DNA polymer.
  • DNA probes For convenience of description reference below to DNA probes should be taken to refer also to RNA probes.
  • a single-stranded sequence of a DNA is built up using known units in a known sequence, at least one such being biotin-linked. The sequence still undergoes the classical hybridisation reaction with a complementary strand of DNA to form a double-stranded DNA. It can thus be incorporated into synthetic DNA probes.
  • any remaining unbound probe must be separated from the bound probe .
  • This separation is normally done by performing the hybridisation reaction in conditions under which sample DNA including the target sequence is immobilized on a substrate and following the binding reaction by washing, or centrifugation to remove any excess probe.
  • the bound probe is therefore all that remains, as an indication or measure of the target sequence. It is detected by the addition of avidin, or streptavidin,to which a marker has been attached.
  • the marker is an enzyme and is for example horse radish peroxidase or alkaline phosphatase.
  • the marker enzyme is then "developed" by reaction with a substrate which produces .a coloured, insoluble precipitate on the membrane surface; the extent of the marker enzyme reaction is usually estimated by eye, or more rarely by densitometry, and although this method provides sensitivity down to the picogram level (Leary et al Proc. Natl. Acad. Sci. USA
  • Our present invention involves a variant on such method of marking the probe sequence, which can also utilise the technology described in our copending European Patent Application 82305597, relating to methods of assay in which a mediator compound is used to transfer charge from an enzyme to an electrode.
  • a mediator compound is used to transfer charge from an enzyme to an electrode.
  • Such a system as therein described may be employed to detect the concentration of either a substrate or an enzyme in a sample, by electrochemical measurement of the occurrence or extent of an enzyme-affected reaction.
  • our copending European Patent Application 84.303090 discloses a novel route to the measurement of nucleic acid probe sequence coupling by using a method developed from the above in which at least one of a mediator and an enzyme, for use in such electrochemical assay are chemically linked to a nucleic acid probe sequence and where any specific binding, of the probe sequence, to the target sequence which may take place thus affects the electrochemical availability of the chemically linked species, for detection by a sensor electrode in presence of the enzyme substrate.
  • Such technology is also relevant to a consideration of the present invention.
  • the present inventio sets out to provide materials, techniques and apparatus to improve the ease of separation in such assays.
  • an entity including a single or double-stranded nucleic acid sequence linked to a magnetic or magnetisable substance.
  • the invention is concerned with a single-strand sequence linked to a magnetic or magnetisable substance, the sequence being a DNA or RNA sequence in a pure or impure form, as obtainable from a natural or a synthetic source.
  • Pure and known sequences will generally comprise a "probe" sequence, but of course a target sequence could be (and usually is) linked to the magnetic or magnetisable substance in accordance with the present invention.
  • the hybridised or other double stranded DNA (RNA) magnetic/magnetisable entity can also constitute a feature of the invention.
  • the magnetic/magnetisable substance is usually provided in a divided or particulate form.
  • a magnetic or magnetisable particle is bound to the target nucleic acid sequence.
  • a substance having a permanent magnetic moment i.e. a ferromagnetic material, especially iron particles or ferrite particles or magnetite particles, Fe 0 , is treated in such a way that the sequence will subsequently attach thereto.
  • this attachment can be effected by simple adsorption or by chemical cross-linking directly to the magnetic material, but a more preferable product has a coating on the magnetic substance of a material to which a nucleic acid material can become permanently attached, for example nitrocellulose.and a nucleic acid attached to this coating .
  • Nucleic acids attached to nitrocellulose are known in the chemical art.
  • the invention also extends to the use of particles of a magnetic or magnetisable substance, coated with a material capable of attachment to a DNA or RNA single-strand material, in a magnetic separation under the influence of an applied magnetic field for the 8 purpose of separating (a) such nucleic acids from a mixture of materials or (b) attached nucleic acids hybridised with a nucleic acid containing a complementary sequence from excess of the unattached complementary nucleic acid or (c) such attached and hybridised materials from an excess of an enzyme marker, or of a material convertible to an enzyme marker, reactive therewith.
  • a method of separation of single-stranded nucleic acid materials from a complex mixture containing such materials in which the " mixture is treated with magnetic or magnetisable material in the form of particles having a coating to which a nucleic acid single strand material becomes permanently attached, and the particles and attached nucleic acid materials are thereafter separated from the other components, at least in part, by a magnetic field.
  • the coating may be nitrocellulose, but cellulose itself (which generally provides functional groups which can be readily derivatised allowing stable covalent attachment of nucleic acids) can be used.
  • a method for the detection or quantification of the presence of a target sequence of nucleic acid units by contact with a probe DNA or RNA sequence of a predetermined level of homology to the target sequences, followed by separation of the materials for detection or quantification of the probe/target hybridisation reaction; in which the probe sequence or the target sequence is a single-stranded nucleic acid sequence linked to a magnetic or magnetisable material in the form of particles having a coating to which a nucleic acid single strand material becomes permanently attached, and. in which separation is at least in part effected by a magnetic field.
  • the present invention is particularly concerned with a method for the detection or quantification of the presence of a target sequence of nucleic acid units, or number of said sequences, in a single-stranded RNA or DNA material, or mixture of such materials, in the presence of a complex liquid mixture containing inter alia such DNA or RNA single-stranded materials, comprising the steps of: (i) contacting the complex liquid mixture with a magnetic or magnetisable material in the form of particles having a coating to which any nucleic acid single-strand RNA or DNA material becomes permanently attached, and thereafter separating the 10 magnetic particles from the mixture, at least in part, by a magnetic field,
  • the probe may be isolated from an organism, or be chemically synthesised, or be synthesised using a host organism.
  • Assay of the probe-target bound species can be effected by known methods.
  • the probe can be chemically linked with compound A of a pair of compounds A and B which themselves react with a specific binding reaction thus useful for measuring probe level in the separated magnetic materials.
  • a and B are antigen/antibody; hormone/receptor; lectin/carbohydrate; or cofactor and enzyme all of which give a known and measurable type of assay for the probe content.
  • a 11 preferred pair of materials A and B are however biotin and avidin, or possibly biotin and streptavidin; the reaction is well known in this context and measurement methods involving fluorescence or enzyme reactions (of an enzyme linked to the avidin) are well-established.
  • biotin/avidin. or biotin/streptavidin specific binding system involves attaching biotin to the probe and a suitable e.g. redox enzyme to the avidin or streptravidin.On addition of suitable substrate a product is formed . The extent or occurrence of this reaction can be measured at an electrode directly or conveyed to an electrode by a suitable mediator such as a metallocene, more especially a ferrocene as disclosed in our earlier Applications itemised above.
  • a suitable mediator such as a metallocene, more especially a ferrocene as disclosed in our earlier Applications itemised above.
  • the enzyme can be glucose oxidase, or glucose dehydrogenase, for use with glucose as a substrate.
  • the preferred enzyme is however a peroxidase e.g. horseradish peroxidase, for use with H O as a substrate. More generally speaking, any redox enzyme can be measured, e.g. in addition to the above, dehydrogenases or those enzymes capable of' attacking or degrading large molecules e.g. ⁇ -amylase, RNA-ases or DNA-ases. 12
  • Ferrocenes bis cyclopentadienyl iron and its derivatives
  • the enzyme of particular preference is glucose oxidase although the enzyme horse radish peroxidase may also be used.
  • the mediator of preference is ferrocene or a derivative thereof. Suitable protocols for the measurement of glucose oxidase concentration by an 13 electrochemical method in the presence of ferrocene are given in the above referenced patent applications.
  • oxidoreductases As described in the patent applications, identified above.it is possible to detect the presence of oxidoreductases using amperometry or coulometry. by coupling the electron transfer from the substrate to the enzyme and thence to a sensor electrode. Such an electrode is generally referred to as an "electron transfer electrode”.
  • a method for detection of a target sequence of nucleic acid units in a single stranded DNA or RNA material, or mixture of such materials, in a liquid mixture comprising the steps of: (i) contacting the liquid mixture with particles of magnetic material (MAG) coated with a celluose derivatives (DCEL) such as nitrocellactose (NITC) whereby single-stranded nucleic acids (NUCA) become permanently attached thereto, and thereafter separating the magnetic particles from the mixture by a magnetic field,
  • the preferred particle coating is cellulose
  • the enzyme is an alkaline phosphatase, capable of producing phenol, for oxidation at a working electrode, from a phenyl phosphate substrate.
  • Such an enzyme can be itself linked by biotin to the avidin (or streptavidin) after the biotin-avidin reaction on the hybridised material.
  • a method for the detection of a target sequence of nucleic acid units in a single stranded DNA or RNA material, or mixture of such materials, in a liquid mixture comprising the steps of:
  • the cellulose derivative (DCEL) can be an aminophenylthioether linkage, activated to the corresponding diazo compound for immobilisation of the single-strand " (NUCA) .
  • the (AV) group can be itself - biotin-linked to the further enzyme, which in one preferred embodiment is an alkaline phosphatase utilising phenyl phosphate as a substrate convertible to phenol, or other substrates. In such a case direct, unmediated, measurement is possible.
  • the invention further extends to apparatus for use in the method of assay as defined above comprising an upper vessel, a lower vessel selectively placeable in liquid flow communication therewith, and selectively applicable magnetic element in said upper vessel.
  • the magnetic element is a magnetisable, or magnetic, grid element having two layers relatively movable to provide either separation or communication between the vessels.
  • the magnetic element may be an electrode for subsequent use in the electrochemical determination of the enzyme reaction; if so it has the potential advantage of drawing towards itself the magnetic species.
  • Figure 1 diagrammatically illustrates steps of an assay method according to the present invention.
  • Figures 2a and 2b show features of an apparatus according to the present invention,generally in vertical cross-section.
  • Figure 3 in its three component parts, shows diagrammatically steps in a method of assay of the present invention carried out in apparatus according to the present invention.
  • Figure 4 in its three component parts, shows variant electrode configurations which can be employed in the method of assay of the present invention.
  • Figure 5 is a graph of concentration in attomoles of ⁇ -DNA. immobilised by hybridisation with a target DNA attached to a cellulose-coated magnetic particulate substrate and labelled with an alkaline phosphatase to produce phenol from a phenyl phosphatase substrate against current in micro-amps at a pyrolytic graphite electrode.
  • step 1 shows the addition of a nitrocellulose-treated magnetic particles (MAG) (NITC) to a mixture containing single stranded nucleic acids (NUCA) to form a complex (MAG)-(NITC)-(NUCA) .
  • MAG nitrocellulose-treated magnetic particles
  • NUCA single stranded nucleic acids
  • An optional magnetic separation can now take place (as described in more detail below) whereby non-DNA or non-RNA material can be separated from the mixture. This leaves a magnetic particle complex of various nucleic acids to be investigated for the target sequence.
  • Step 2 shows addition of excess of a probe sequence linked to biotin.
  • which by a hybridisation reaction forms the complex (MAG)-(NITC)-(NUCA)-(PRO)-(BIO) with those nucleic acids containing the target sequence, leaving excess (PRO) - (BIO) unreacted.
  • a magnetic field is applied to retain all complexes containing the (MAG) and therefore those complexes including the target nucleic acid.
  • the excess (PRO)-(BIO) can therefore be washed away without substantial loss of the complex of probe and target nucleic acid.
  • the other (NUCA) species are still present, but not labelled with (PRO)-(BIO).
  • Step 3 shows addition of excess of a detector system for the biotin marker employed.
  • the detector system in this particle example comprises a conjugate of avidin or streptavidin (AV) and horseradish peroxidase (PER), although other systems can of course be used.
  • AV avidin or streptavidin
  • PER horseradish peroxidase
  • the system comprises (MAG)-(NITC)-(NUCA)-(PRO)- (BIO)-(AV)-(PER) plus excess (AV)-(PER).
  • a magnetic field is applied to retain the large complex and the excess (AV)-(PER) is washed away without disturbing the large complex.
  • the assay mixture will still contain much or all of the original nucleic acid which was absorbed onto the coated magnetic particles.
  • only those target sequences which are homologous to the probe have formed a duplex with the probe and have therefore complexed the peroxidase.
  • the concentration of peroxidase in the solution is related directly to the degree of duplex formation and therefore to the degree of homology between the original target nucleic acid and the probe.
  • Step 4 shows an electrode (E) placed in contact with the resulting suspension containing (MAG)-(NITC)-(NUCA)-(PRO)-(BIO)-(AV)-(PER) .
  • the H O the substrate of the peroxidase, is added together with ferrocene as a suitable mediator compound.
  • the catalytic current generated is proportional to the (AV)-(PER) activity and hence to the concentration of the nucleic acid which has been hybridised to the probe.
  • the assay mixture still contains much of the original nucleic acid (in a partially purified form) it is possible to probe the assay mixture again for other sequences, by the use of other probes. Unless these additional probes bind to sequences which overlap with those which have already formed a duplex, further hybridisation can occur between the target nucleic acid and the new probe and the analysis may be continued (with a suitable compensation for the background peroxidase activity) .
  • Figure 2 shows one form of an apparatus according to the present invention and for use in the method of the present invention. It consists of two vessels (1 and 2) separated by a magnetic filter (3). Flanged edges (4 and 5) are provided to seal the joint between the vessels.
  • the filter comprises a movable magnetic or magnetisable grid or gauze (6) together with a fixed gauze (7) which latter for ease of operation should preferably be coated with a teflon_schreib or
  • the magnetic gauze (6) can if necessary be used as an electrode; if so it should have a surface of gold or other suitable metal, electroplated or otherwise deposited onto a magnetisable or magnetic gauze support. If the grid or gauze is not to be used as an electrode, it can be coated with a material such as teflon and silanised to inhibit absorption. The interior of the vessels should also be silanised before use or made of a suitable material such as polypropylene to prevent absorbtion of materials on to the vessel walls.
  • the lower vessel (2) is provided with a drain (16) and a suction port (15) .
  • the grid or gauze can be constructed of any permanent magnetic material, such as iron, cobalt or nickel. Alternatively, if windings are placed around the joint, a potentially magnetisable material can become magnetic on the passage of a current and demagnetised when the current is off.
  • the flanged edges (4 and 5) may be greased, or provided with O-rings (9) or both. Alternatively, a bayonet-type fitting may be employed.
  • An exterior holding clip or clamp (8) may be provided to ensure a good seal and/or rigidity of the apparatus.
  • the upper grid (6) is movable relative to the lower grid by a sliding action.
  • the upper grid is shown to be moved by a tag (10).
  • a number of other methods can be used to provide movement such as a rod along the axis of the upper vessel.
  • the tag is advantageous in that it may form an electrical terminal if the upper grid (6) is to constitute an electrode.
  • FIGs 2a and 2b Further detail of the sliding action is shown in Figures 2a and 2b.
  • the two grids are open to the passage of materials in Figure 2a but closed to the passage of materials in Figure 2b.
  • a coil (11) is located about the lower part of the upper vessel and generates a magnetic field when current is passed through it.
  • a permanent magnet may be employed, either hand-held or as part of a test-tube rack) .
  • Fig. 3 shows steps of a method of the present invention carried out in an apparatus according to the present invention.
  • a magnetic field is applied to the apparatus by the coil (11).
  • Ferromagnetic members (13) may if desired be placed within the field generated by the coil (11) so that the magnetic particles (12) become immobilised upon them and are held in the upper vessel (11).
  • a magnetic or magnetisable grid is present the magnetic particles (12) attach themselves to this grid when the field is on.
  • the grid is then opened to the passage of materials from the upper vessel to the lower vessel.
  • the assay mixture may therefore be washed through by the addition of a suitable fluid from the direction indicated by the letter A in the figure. Excess (PRO)-(BIO) therefore becomes washed out of the apparatus in the direction indicated by the letter B.
  • the coil (11) may if desired also be employed to agitate the magnetic particles within the upper chamber by the application of a suitably varying magnetic field.
  • Fig. 4 shows in its three parts and in diagrammatic cross-sections three different electrode configurations suitable for use with the apparatus of the present invention in the final stage of direct electrochemical measurement of the enzyme reaction.
  • Fig. 4a shows a more or less orthodox three-electrode configuration comprising a working electrode (W) , an auxiliary electrode (A) and a reference electrode (R) . Further details and examples of electrode configurations of this general type and of a similar, two-electrode, type are given in the patent applications identified above.
  • Fig. 4b shows a working electrode (W) provided with a winding (14) to enable the electrode itself to become magnetic as and when desired. This has the effect of concentrating on the electrode reagents which are per se magnetic or magnetisable.
  • Fig. 4c shows the working electrode (W) constitute as one or other component of the grid.
  • the following example shows stages in the preparation and properties of a particularly valuable ferrocene derivative which can be utilised in one preferred electrochemical measurement procedure, and which exemplify another aspect of the present invention which consists in a nucleotide labelled with a covalently-linked electoactive species, e.g. a metallocene such as ferrocene; more specifically a ferrocene-labelled -UTP is envisaged.
  • Another aspect of the invention comprises the use of such materials in the assay of nucleic acid sequences.
  • Example 2 PART I Preparation of 5-Ferrocenyl Pentanoi ⁇ Acid
  • Tetrahydrofuran (THF) 100 mL
  • Fc(CH ) COC1 hexane solution of Fc(CH ) COC1.
  • the solution was filtered to remove an offwhite precipitate. Removal of volatiles under vacuum gave the title compound as a yellow powdery solid.
  • the product was further purified by column chromatography on silica with acetone-ether (1:1) as eluant.
  • the desired compound eluted as a major yellow band after two minor yellow components and forms an orange oil on removal of volatiles, which subsequently crystallised to a yellow solid on standing at room temperature. This was identified as ferrocenylated 2'-3' isopropylidene uridine conjugate. Melting point 60-62°C
  • This example shows a variant procedure and use of a different electrochemical measurement system.
  • ⁇ DNA was biotinylated (to provide a probe material) using nick-translation kit (purchased from BRL Ltd) in the presence of biotin-11-UTP.
  • High molecular weight DNA was purified from unincorporated material by chromatography on a Nensorb cartridge (Dupont). The DNA solution was evaporated to dryness and the pellet was reconstituted in lxSSPE buffer at 37 for 20 min.
  • Hybridisations were carried out in polypropylene vials, sealed in polypropylene bags, submerged in a shaking water bath. ⁇ DNA samples linked to the magnetic support were pre-incubated at 65 C for two hours in a cocktail which consisted of 4 x SSPE. 6 x Denhardt's solution, 300 ⁇ g/ml denatured salmon DNA and 0.1% W /v SDS (1 ml).
  • the pre-hybridisation mix was removed and replaced with 1 ml of hybridisation cocktail consisting of 4 x SSPE, 2 x Denhardt's solution. 200 ⁇ g/ml denatured salmon sperm DNA. 0.1% w /v SDS, 10% w /v dextran sulphate in the following buffers:
  • the magnetic particles were washed at 22°C for 90 min in 1 ml blocking buffer (100 M NaCl, 100 mM Tris/HCl. 3 mM MgCl 2 . 0.5% V /v Tween 20, at pH 7.5), magnetic separation as before also being utilised as a separation technique.
  • 1 ml blocking buffer 100 M NaCl, 100 mM Tris/HCl. 3 mM MgCl 2 . 0.5% V /v Tween 20, at pH 7.5
  • Alkaline phosphatase bound to the magnetic matrix was finally detected electrochemically by resuspending the supporting material in 1 ml of developing buffer which contained 5 mM phenyl phosphate, and incubating at 22°C for 10 min. The supernatant was decanted and transferred to a standard three electrode cell (saturated calomel reference, platinum counter and pyrolyti ⁇ graphite working electrode). Phenol produced by the action of alkaline phosphatase was oxidised at the working electrode which was poised at +600 mV, and the resulting current versus time transients were recorded.
  • Figure 5 shows the currents obtained when magnetic particles containing various amount of immobilised DNA were probed using the protocols described in the methods section.
  • the results demonstrate the sensitivity of the detection system employed in that 1 attomole of DNA was readily detected above background levels. Furthermore, in the range examined, the current response is directly proportional to the amount of immobilised DNA on the magnetic support.
  • the simple one step hybridisation protocol described above thus has the potential to detect DNA sequences present at the single copy level in mammalian DNA and to allow the quantitation of multiple copy gene sequences.
  • the system can be extended and modified to produce further configurations, for example; (a) Any marker enzymes that can be either biotinylated and/or covalently linked to streptavidin can be used provided that they produce electrochemically active products (i.e. glucose oxidase. horse radish peroxidase) .

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Particules magnétiques revêtues de dérivés de cellulose, par exemple la nitrocellulose, s'unissant de manière à former un support pour les séquences d'ADN ou d'ARN. Une séquence sonde, elle même marquée, par exemple avec de la biotine, s'hybride avec des séquences supportées complémentaires appropriées, et les complexes résultant sont séparés magnétiquement. L'avidine, ou la streptavidine, s'accouple ensuite avec la biotine et le complexe est de nouveau séparé magnétiquement. L'avidine peut être déjà liée avec un enzyme marqueur tel que le péroxyde de raifort, ou la glucose oxydase, ou la phosphatase alcaline, ou peut être par la suite liée à ces substances. Dans les deux cas, un contact ultérieur avec un substrat approprié à l'enzyme (H2O2, glucose, phosphate de phényle) produit une réaction permettant une mesure électrochimique soit via un composé médiateur de ferrocène (H2O2, glucose soit par oxydation sur la surface d'une électrode (phénol à partir de phosphate de phényle). Cette réaction est influencée de manière très sensible par la présence d'une hybridation initiale ou par la quantité de cette hybridation; des quantités d'attomole sont mesurables.
PCT/GB1986/000174 1985-03-25 1986-03-25 Sequences d'acide nucleique liees a des materiaux sensibles a des champs magnetiques, et methodes d'analyse et appareils utilisant lesdites sequences liees WO1986005815A1 (fr)

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GB858507706A GB8507706D0 (en) 1985-03-25 1985-03-25 Magnetic nucleic acid sequences
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EP0265244A3 (en) * 1986-10-23 1989-05-10 Amoco Corporation Target and background capture methods and apparatus for affinity assays
EP0288737A1 (fr) * 1987-04-01 1988-11-02 Miles Inc. Essai d'hybridation rapide utilisant une sonde immobilisée sur latex
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US6013531A (en) * 1987-10-26 2000-01-11 Dade International Inc. Method to use fluorescent magnetic polymer particles as markers in an immunoassay
EP0328829A3 (en) * 1987-12-21 1990-09-19 Amoco Corporation Method for affinity assays using target amplification
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WO1990002205A1 (fr) * 1988-08-25 1990-03-08 Angenics, Inc. Detection de sequences d'acides nucleiques par agglutination de particules
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EP0371437A3 (fr) * 1988-11-29 1991-03-27 Orion-Yhtymà„ Oy Procédé et combinaison de réactif pour déterminer des séquences nucléotides
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EP0679891A3 (fr) * 1989-11-17 1995-11-29 Merck Clevenot Lab
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WO1991015768A1 (fr) * 1990-04-06 1991-10-17 Syngene, Inc. Methode et composition destines aux tests d'adn
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