WO2000022165A1 - Improved hybridisation assay in which excess probe is destroyed - Google Patents
Improved hybridisation assay in which excess probe is destroyed Download PDFInfo
- Publication number
- WO2000022165A1 WO2000022165A1 PCT/GB1999/003383 GB9903383W WO0022165A1 WO 2000022165 A1 WO2000022165 A1 WO 2000022165A1 GB 9903383 W GB9903383 W GB 9903383W WO 0022165 A1 WO0022165 A1 WO 0022165A1
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- Prior art keywords
- nucleic acid
- nuclease
- hybrid
- reagent
- antibody
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Classifications
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- 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
- C12Q1/6832—Enhancement of hybridisation reaction
-
- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
Definitions
- This invention relates to methods for detecting nucleic acids.
- Nucleic acid hybridisation is a widely used technique for identifying, detecting and quantitating target polynucleotide sequences in a sample. This technique relies for its success on complementary base pairing between the two halves of a double-stranded nucleic acid molecule: when single-stranded nucleic acids are incubated in solution under suitable conditions of temperature, pH and ionic strength, complementary base sequences pair to form double-stranded stable hybrid molecules. This ability of single-stranded nucleic acid molecules to form a hydrogen-bonded structure with their complementary nucleic acid sequences has long been employed as an analytical tool in recombinant DNA research.
- the sample will contain double-stranded nucleic acid and must be denatured prior to the hybridisation assay to render it single-stranded.
- a nucleic acid having a known sequence which is complementary to the target sequence is either synthesised chemically in an automated fashion with great facility, or is isolated from the appropriate organism and rendered single-stranded by denaturation. It is then used as a probe to search a sample for a target complementary sequence. Detection of specific target nucleic acids enables accurate diagnosis of bacterial, fungal and viral disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence enables the diagnosis of human genetic disorders. Hybridisation produces stable hybrids, and a number of different approaches are known to the art for detecting these.
- labelled probes By labelling a probe nucleic acid with some readily detectable chemical group, it is possible to detect the polynucleotide sequence of interest in a test medium containing sample nucleic acids in single-stranded form. Nucleic acids have been labelled with radioisotopes, enzymes and fluorescent molecules. The use of labelled nucleic acids as probes in macromolecular analysis is important for clinical, veterinary and environmental diagnostic applications.
- the amplification system comprises an apoenzyme which is convertible into a holoenzyme by interaction with an accessory subunit and a masked form of the subunit which is convertible into its active unmasked form by the action of the enzyme to be detected.
- a hydrolase enzyme able to hydrolyse a synthetic derivative of FAD substituted in such a way that it yields FAD when hydrolysed, and is incorporated herein by reference in its entirety.
- the subunit is FAD and the masked form is 3'FADP
- the apoenzyme is apo-glucose oxidase or apo-D-aminoacid oxidase.
- the FAD produced forms an active holoenzyme from the corresponding apoenzyme.
- This approach allows the detection of small amounts of alkaline phosphatase in short periods of time. For example, using such an amplification system in which the apoenzyme is apo-D-amino acid oxidase has permitted the detection of OJ amol of alkaline phosphatase in less than 30 minutes (Harbron et al., Anal. Biochem. (1992) 206: 119 - 124).
- this approach is further extended to an amplification assay for nuclease P
- DNA or RNA is amplified by the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- This method involves the hybridisation of an oligonucleotide primer to the 5' end of each complementary strand of the double- stranded target nucleic acid.
- the primers are extended from the 3' end in a 5' ⁇ 3' direction by a DNA polymerase which incorporates free nucleotides into a nucleic acid sequence complementary to each strand of the target nucleic acid.
- the extension products become target sequences for the next cycle.
- repeated cycles must be carried out, between which cycles, the complementary DNA strands must be denatured under elevated temperatures.
- ligase chain reaction A method of detecting a specific nucleic acid sequence present in low copy in a mixture of nucleic acids, called ligase chain reaction (LCR), has also been described.
- LCR ligase chain reaction
- WO 89/09835 describes this method and is incorporated herein by reference in its entirety.
- Target nucleic acid in a sample is annealed to probes containing contiguous sequences.
- the probes ligated to form detectable fused probes complementary to the original target nucleic acid.
- the fused probes are disassociated from the nucleic acid and serve as a template for further hybridisation's and fusions of the probes, thus amplifying geometrically the nucleic acid to be detected.
- the method does not use DNA polymerase.
- nucleic acid amplification procedures include transcription- based amplification systems (Kwoh et al., Proc. Natl. Acad. Sci. (U.S.A.) (1989) 86:1173; Gingeras et al., WO 88/10315; Davey et al., EP 329,822; Miller et a/., WO 89/06700), RACE (Frohman, In: PCR Protocols: A Guide to Methods and Applications, Academic Press, NY (1990)) and one-sided PCR (Ohara, et al, Proc. Natl. Acad. Sci. (U.S.A.) (1989) 86:5673-5677).
- Particularly suitable amplification procedures include Nucleic Acid Sequence-Based Amplification, Strand Displacement Amplification, and Cycling Probe Amplification.
- Target amplification techniques described in the foregoing are generally complex and susceptible to inhibition, contamination and amplification of the wrong target.
- the somewhat simpler approach of sandwich hybridisation therefore remains attractive.
- the reporter probe is a nucleic acid having a sequence complementary to at least part of the target sequence and which is labelled with a detectable group.
- the capture probe is a nucleic acid having a sequence complementary to at least part of the target sequence, but which is different to that of the reporter probe, and which is labelled with an immobilisable group.
- pairs of specific binding members sbm's have been used for this purpose.
- Disadvantages of these approaches include the increased cost and complexity of using two probes. For example, for each assay two probes need to be synthesised and labelled: one for use as the capture probe, and the other for use as a reporter probe. In addition, hybridisation conditions have to be carefully chosen to form the sandwich of target, capture probe and reporter probe.
- Atlas and Steffan disclose a solution hybridisation method for detecting genetically-engineered micro-organisms in environmental samples.
- the detection method involves recovery of DNA from the microbial community of an environmental sample followed by hybridisation in solution with a radio-labelled RNA gene probe. After nuclease digestion of non- hybridised probe RNA, the DNA-RNA hybrids formed in the solution hybridisation are separated by column chromatography and detected by liquid scintillation counting.
- JP04135498A A similar approach is disclosed in JP04135498A in which hybrids are treated to remove any single-stranded material present prior to purification of the hybrid by gel filtration chromatography, with subsequent detection by non- radioactive techniques.
- Resulting hybrids are detected by binding of an antibody reagent, preferably labelled with a detectable chemical group, selective for binding the hybrids in the presence of the single-stranded sample and probe nucleic acids.
- an antibody reagent preferably labelled with a detectable chemical group
- Resulting hybrids are detected by binding of an antibody reagent, preferably labelled with a detectable chemical group, selective for binding the hybrids in the presence of the single-stranded sample and probe nucleic acids.
- Carrico's invention is the requirement for antibodies specific for double-stranded hybrids having little affinity for single-stranded nucleic acid.
- the generation of specific polyclonal antibodies that will bind double-stranded nucleic acid but not single-stranded nucleic acid is complicated by the fact that polyclonal antisera may contain antibodies that will cross-react with single-stranded nucleic acid.
- Polyclonal antisera may also contain naturally occurring antibodies to single-stranded nucleic acid or antibodies to single-stranded nucleic acid arising as a result of the immunisation.
- Monoclonal antibody technology can provide a means to select an antibody with desired affinity and specificity which will overcome in part these problems.
- Pat. No. 5612,458 to Hyldig-Nielson and Pluzek use antibodies to complexes between peptide nucleic acid (PNA) and nucleic acids, particularly antibodies to nucleic acid probe-DNA or nucleic acid probe-RNA hybrids.
- PNA peptide nucleic acid
- a still further approach to eliminating problems associated with potential cross-reactivity of antibodies used in these assays is to use a probe labelled with one member of a specific binding pair, which can be captured with the second member of the pair after hybridisation and treatment with a nuclease.
- This approach is disclosed in EP0780479A. But again, this suffers from the inability of the endonuclease digestion approach (see above) to efficiently separate hybridised from unhybridised molecules, and problems associated with contamination of the hybridisation mixture with a nuclease, also noted above.
- a nucleic acid probe which comprises a sequence complementary to a target nucleic acid and an enzyme reagent able to hydrolyse single-stranded nucleic acid, but which is substantially without effect on double-stranded nucleic acid.
- an enzyme reagent able to hydrolyse single-stranded nucleic acid, but which is substantially without effect on double-stranded nucleic acid.
- the pH is adjusted to a value within the activity range of the enzyme reagent, and single stranded nucleic acid, including unhybridised probe, is hydrolysed.
- the attachment of the enzyme reagent directly to the probe facilitates hydrolysis of unhybridised probe and sterically hinders hydrolysis of hybrids, which overcomes the inability of the endonuclease digestion approach used by Atlas and Stefan and others to efficiently separate hybridised from unhybridised molecules, as observed by Fliss et al.
- the hybrid may be captured by an agent, which may be an antibody or nucleic acid binding protein specific for the double-stranded hybrid, or it may be one member of a pair of specific binding members, the other member attached to the hybrid.
- the agent itself may be immobilised or immobilisable.
- the nucleic acid probe may itself be immobilised or immobilisable.
- the present invention discloses a new and improved method for detecting single-stranded target nucleic acid.
- a method for detecting a single-stranded target nucleic acid comprising the steps of:
- step (c) characterised by, prior to step (c), bringing the nucleic acid probe or hybrid into contact with a solid support to attach it thereto or bringing the nucleic acid probe or hybrid into contact with a capture reagent, optionally linked to a solid support, to capture the nucleic acid probe or hybrid; and washing the capture reagent or solid support on which the hybrid is immobilised with a washing fluid while the capture reagent or solid support is contained within a vessel that is adapted to retain the capture reagent or solid support but not to retain fluid in which the capture reagent or solid support is dispersed, whereby material which has not been captured by the capture reagent or otherwise immobilised on a solid support is eluted from the vessel.
- the method comprises the steps of: (a) forming a hybrid between a target nucleic acid and a nucleic acid probe, said nucleic acid probe labelled with an enzyme reagent which hydrolyses single-stranded nucleic acid but is substantially without effect on double-stranded nucleic acid, said hybrid formed under conditions of pH which are outside the activity range of said enzyme reagent,
- nucleic acid probe labelled with an enzyme reagent which hydrolyses single-stranded nucleic acid but is substantially without effect on double-stranded nucleic acid, said hybrid formed under conditions of pH which are outside the activity range of said enzyme reagent, said nucleic acid probe attached to a solid support contained in a vessel able to retain the support but not able to retain a solution in which said support is dispersed, b) adjusting said pH to a value within the activity range of said enzyme reagent, whereby said enzyme reagent substantially hydrolyses any single-stranded nucleic acid present,
- the invention provides a variety of detection means for detecting the hybrid.
- the detection means may be a hybrid-binding reagent such as an antibody or DNA-binding protein specific for double-stranded nucleic acid.
- the detection means may also be a pair or pairs of specific binding members. These may be an antigen or hapten and the corresponding antibody; biotin and avidin, streptavidin or neutravidin; or a nucleic acid binding protein specific for a sequence present in the nucleic acid probe. Any of these agents may be labelled with a detectable label, which may an enzyme, a fluorescent moiety, a chemiluminescent moiety, an electro-chemiluminescent moiety or a coloured moiety.
- the invention provides a variety of capture means.
- the capture means may be a hybrid-binding reagent such as an antibody or DNA- binding protein specific for double-stranded nucleic acid.
- the capture means may also comprise one member of a pair or pairs of specific binding members. These may be an antigen or hapten and the corresponding antibody; biotin and avidin, streptavidin or neutravidin; or a nucleic acid binding protein specific for a sequence present in the nucleic acid probe. These agents are attached to an insoluble support either directly or indirectly by means of an immobilisable label.
- the capture means may also be a material which binds nucleic acids relatively non-specifically, including silica materials, ion-exchange materials, hydrophobic materials, materials used in reversed-phase chromatography, and the like.
- the invention provides a variety of support means. These include agaroses and their derivatives, polyacrylamides and their derivatives, magnetic materials, cellulosic materials, acrylic materials and the like.
- the invention discloses a method for detecting DNA-RNA hybrids, DNA-DNA, RNA-RNA, DNA-RNA or DNA-PNA hybrids between a target nucleic acid and a nucleic acid probe having a sequence complementary to part of the target nucleic acid.
- the invention discloses a method for detecting hybrids between nucleic acid amplification products and a nucleic acid probe having a sequence complementary to part of the amplified nucleic acid.
- the invention discloses a method for detecting hybrids between target nucleic acid extracted from a clinical specimen, a veterinary specimen, a food specimen or an environmental sample and a nucleic acid probe having a sequence complementary to part of the target nucleic acid.
- the invention provides a kit for carrying out the method.
- Advantages of the present invention are: only a single probe is required; efficient washing is achieved using a column format; the method may be easily and advantageously automated; highly sensitive detection systems, such as chemiluminescence or enzyme amplification cascades may be used to detect the hybrids; and the sensitive detection of target nucleic acid may be achieved without using target amplification techniques, such as PCR or LCR.
- a single type of capture agent attached to a column material may be used for any analyte; an advantage of other embodiments is that a single detection reagent may be used, (d) to provide a method for detecting hybrids between DNA-RNA, RNA- DNA, RNA-RNA, RNA-PNA and DNA-PNA hybrids by appropriate selection of the hybrid binding reagent and enzyme reagent used.
- the vessel in which the hybrid is retained in the method is preferably a column and suitably of the type used for column chromatography or it may, for example, comprise a syringe or other open ended vessel with a filter or other means for retaining the hybrid that is immobilised on the capture reagent or solid support during the washing step.
- Figure 1 is a diagrammatic representation of three preferred embodiments of the present invention for the detection of single-stranded nucleic acids.
- Figure 2 shows a standard curve for the 3'FADP-based enzyme amplification assay of nuclease P, (filled triangles) and nuclease S, (filled squares).
- the abscissa represents the amount of each enzyme present in amol (10 "18 mol), and the ordinate represents the absorbance obtained after 15 mins incubation at 25° C after subtraction of the blank reading. Both scales are logarithmic.
- the dotted line represents the detection limit.
- the present invention provides a column-based method for detecting hybrids formed between a target nucleic acid and a nucleic acid probe.
- the probe is labelled with (ie has joined to its nucleic acid sequence) an enzyme reagent specific for single-stranded nucleic acids, which hydrolyses all unhybridised single-stranded nucleic acids present: this means that the hybridisation assay may be performed using only one probe.
- the probe is attached to a column material; in another, a capture reagent is attached to a column material. In both cases, the use of a column format increases the efficiency of the washing steps needed to remove hydrolysed materials from the column.
- the target nucleic acid may be DNA or RNA, and is obtained from any medium of interest, for example, a liquid sample of medical, veterinary, environmental, or industrial significance.
- the target nucleic acid may also be the product of a nucleic acid amplification assay, such as PCR or LCR. If the target nucleic acid is principally double stranded, it will be treated to denature it prior to the formation of the hybrid. Denaturation of nucleic acids is preferably accomplished by heating in boiling water or alkali treatment (e.g., 0J N sodium hydroxide), which if desired, can simultaneously be used to iyse cells.
- alkali treatment e.g., 0J N sodium hydroxide
- release of nucleic acids from cellular or viral sources can, for example, be obtained by mechanical disruption (freeze/thaw, abrasion, sonication), physical/chemical disruption (detergents such as TritonTM, Tween, or sodium dodecylsulfate, alkali treatment, osmotic shock, or heat), or enzymatic lysis (lysozyme, proteinase K, pepsin).
- the resulting test medium will contain the target nucleic acid in single-stranded form.
- the nucleic acid probe may be a DNA probe an RNA probe, or a PNA probe.
- the nucleic acid probe will comprise at least one single-stranded base sequence substantially complementary to at least part of the target nucleic acid sequence.
- base sequence need not be a single continuous polynucleotide segment, but can be comprised of two or more individual segments interrupted by non-complementary sequences. These non-hybridisable sequences are linear.
- the complementary region of the nucleic acid probe can be flanked at the 3'- and 5'-termini by non-hybridisable sequences, such as those comprising the DNA or RNA of a vector into which the complementary sequence had been inserted for propagation.
- the nucleic acid probe as presented as an analytical reagent will exhibit detectable hybridisation at one or more points with target nucleic acids of interest.
- the nucleic acid probe sequence can be of any convenient or desired length, ranging from as few as a dozen to as many as 10,000 bases, and including oligonucleotides having less than about 50 bases.
- the nucleic acid probe may be an oligonucleotide produced by solid-phase chemistry by a nucleic acid synthesiser.
- the RNA or DNA probe can be obtained in a variety of other conventional manners.
- RNA probe and DNA probe it is not implied that all nucleotides comprised in the probe be ribonucleotides or 2'-deoxyribonucleotides. Therefore, one or more of the 2'-positions on the nucleotides comprised in the probe can be chemically modified provided the antibody binding characteristics necessary for performance of the present assay are maintained to a substantial degree.
- the nucleic acid probe can have in general any other modification along its ribose phosphate backbone provided there is no substantial interference with the specificity of the antibody to the double stranded hybridisation product compared to its individual single strands.
- the nucleic acid probe in addition to the enzyme label, is labelled with either a detectable moiety or an immobilisable moiety.
- the nucleic acid probe is prepared by solid-phase chemistry using a nucleic acid synthesiser and has a trityl-hexyl thiol derivatised 5'-end.
- the covalent attachment of the label to this moiety may be achieved by a number of well-known methods using a wide range of heterobifunctional reagents. For example, the method of Carlsson et al.
- the label is reacted with 3-[(2)-pyridyldithio]propionic acid N-hydroxysuccinimide ester (SPDP) to give a 2-pyridyl disulphide-activated label.
- SPDP 3-[(2)-pyridyldithio]propionic acid N-hydroxysuccinimide ester
- This allows disulphide exchange with trityl-hexyl thiol derivatised described above to yield a labelled nucleic acid probe.
- Other approaches for labelling the nucleic acid probe will be apparent to one skilled in the art. Additionally, a wide range of labelled nucleic acids is available from commercial sources.
- Preferred labels include the enzymes alkaline phosphatase, peroxidase, - galactosidase, nuclease P 1 ( nuclease S, and mung bean nuclease; the haptens digoxin, digoxygenin, fluorescein, fluorescein isothiocyanate; and biotin or biotin analogues.
- a preferred embodiment of the present invention employs a nuclease as the enzyme reagent.
- a number of nucleases are known which are specific for single- stranded nucleic acids.
- ribonuclease A and ribonuclease T may be used in combination to hydrolyse single-stranded RNA.
- Other preferred nucleases include exodeoxyribonuclease I (E.C. 3.1.11.1 , similar enzymes: mammalian DNase III, exonuclease IV, T2- and T4-induced exodeoxyribonucleases), exodeoxyribonuclease (phage sp3-induced) (E.C. 3.
- exodeoxyribonuclease V (E.C. 3.1.11.5, similar enzyme: Haemophilus influenzae ATP-dependent DNase), exodeoxyribonuclease VII (E.C. 3.1.11.6, similar enzyme: Micrococcus luteus exonuclease), exoribonuclease II (E.C. 3.1.13.1 , similar enzymes: RNase Q, RNase BN, RNase Pill, RNase Y), venom exonuclease (E.C. 3.1.15.1 , similar enzymes: hog kidney phosphodiesterase, Lactobacillus exonuclease), spleen exonuclease (E.C.
- nuclease S1 E.C. 3.1.30.1
- similar enzymes N crassa nuclease, mung bean nuclease, Penicillium citrinum nuclease P.,
- Particularly preferred nucleases are nuclease P 1t nuclease S, and mung bean nuclease, which have a relatively broad specificity against single- stranded DNA and RNA.
- hybrid-binding reagent is an antibody
- this may be obtained in any available manner such as conventional antiserum and monoclonal techniques.
- Antiserum can be obtained by well-established techniques involving immunisation of an animal, such as a mouse, rabbit, guinea pig or goat, with an appropriate immunogen.
- the immunoglobulins can also be obtained by somatic cell hybridisation techniques, also involving the use of an appropriate immunogen.
- the antibody reagent may also be a recombinant antibody, a chimeric antibody, or a single chain antibody.
- the antibody may be specific for RNA-DNA hybrids, DNA- DNA hybrids or RNA-RNA hybrids.
- An example of the production of anti-DNA-RNA monoclonal antibodies is given by Fliss et al. (Applied and Environmental Microbiology (1993) 59: 2698 - 2705).
- Antibodies specific for double-stranded nucleic acid may also be obtained from commercial sources.
- the antibody is labelled with either a detectable moiety or an immobilisable moiety.
- the covalent attachment of the label may be achieved by a number of well-known methods using a wide range of heterobifunctional reagents. For example, the method of Carlsson et al. (Biochem J (1978) 173: 723 - 737) may be used: the label is reacted with 3-[(2)-pyridyldithio]propionic acid N- hydroxysuccinimide ester (SPDP) to give a 2-pyridyl disulphide-activated label.
- SPDP 3-[(2)-pyridyldithio]propionic acid N- hydroxysuccinimide ester
- Preferred labels include the enzymes alkaline phosphatase, peroxidase, -galactosidase, nuclease P 1 f nuclease S, and mung bean nuclease; the haptens digoxin and digoxigenin, and biotin or biotin analogues.
- a wide range of suitable support materials is commercially available. These are preferably activated to allow the probe or the hybrid-capture agent to be attached. These include N-hydroxy-succinimide activated matrices from Pharmacia Biotech, such as Superose, Sepharose and Hi-Trap materials. N-hydroxysuccinimide- and hydrazide-activated Affigels are available from BioRad. A wide range of activated supports may also be obtained from Sigma, including CN-Br activated, epoxy-activated, nitrophenyl-chloroformate activated, N-hydroxy- succinimidyl chioroformate activated, oxirane activated and polyacryl-hydrazido activated materials; thiolated materials are also available.
- a magnetic support material may also be usefully employed.
- a preferred embodiment includes a retrievable support comprising magnetic beads characterised in their ability to be substantially homogeneously dispersed in a sample medium.
- the magnetic beads contain primary amine functional groups that facilitate covalent binding or association of a probe entity to the magnetic support particles.
- the magnetic support beads are single domain magnets and are super paramagnetic exhibiting no residual magnetism.
- a magnetic bead suitable for application to the present invention includes a magnetic bead containing primary amine functional groups marketed under the trade name BIO-MAG by Advanced Magnetics, Inc. Beads having reactive amine functional groups can be reacted with polynucleotides to covalently affix the polynucleotide to the bead. The beads are reacted with 10 percent glutaraldehyde in sodium phosphate buffer and subsequently reacted in a phosphate buffer with ethylene-diamine adduct of the phosphorylated polynucleotide.
- the support material may itself serve as the hybrid-capture agent.
- the support may be an anionic exchange resin able to bind nucleic acids through a charge interaction. Suitable resins include DEAE-Sephadex, DEAE- Cellulose, a Mono-Q resin and the like.
- the support may also be of glass wool, glass beads or other silicaceous material well known for binding nucleic acids. Other materials able to bind nucleic acids may also be used. Commercially available materials include Biospin columns from BioRad; Nucleon QC, EasyPrep and Microspin columns from Amersham-Pharmacia Biotech; Wizard systems from Promega; and Xtreme kits from Pierce.
- Fig 1 shows the first row shows the target nucleic acid (2), denatured if necessary to render it single-stranded, being contacted under hybridisation conditions with a nucleic acid probe (4) having a sequence complementary to at least part of the target nucleic acid and labelled at its 5'-end with an enzyme reagent (6), preferably nuclease P
- nucleic acid probe (4) is attached to support material (10).
- nucleic acid probe (4) is additionally labelled at its 3'-end with a first member of a specific binding pair (8), preferably biotin.
- the pH of the mixture is adjusted to allow enzyme reagent (6) to remove single-stranded nucleic acids.
- These single-stranded nucleic acids comprise unhybridised probe and unhybridised target.
- the hybrid attached to support material (10), is introduced into a suitable column and washed with a washing agent, preferably TBS-Tween. This elutes hydrolysed materials.
- a washing agent preferably TBS-Tween. This elutes hydrolysed materials.
- the hybrid is detected directly through enzyme reagent (6) or indirectly through a binding agent specific for the hybrid (12).
- the hybrid is captured onto a support material (10), either through a second member of a specific binding pair (14), or through a hybrid-binding agent (12).
- the hybrid-binding agent may be relatively specific for the hybrid, preferably antibody specific for double-stranded DNA, or relatively non-specific, preferably silica.
- the hybrid is detected directly through enzyme reagent (6), indirectly through a second member of a specific binding pair (14), or indirectly through a hybrid-binding agent (12) specific for the hybrid and labelled with a detectable moiety.
- the hybrid is captured onto a support material (10) through a hybrid-binding agent (12), and detected directly through enzyme reagent (6).
- the hybrid-binding agent may be relatively specific for the hybrid, preferably antibody specific for double-stranded DNA, or relatively non-specific, preferably silica.
- nuclease P is shown to be joined directly to the nucleic acid probe.
- the link may also be a n indirect one: for example: embodiments are envisaged in which the probe is labelled with a moiety, such as flourescein isothiocyanate, and nuclease P., is attached thereto by means of an anti-FITC antibody labelled with nuclease P
- a moiety such as flourescein isothiocyanate
- a kit for carrying out the described methods according to the present invention contains a sbm specific for the hybrid or a moiety present on the nucleic acid probe attached to a support material, optionally contained in a column, a nucleic acid probe that is complementary to the target nucleic acid to be detected and which is labelled with an enzyme reagent specific for single-stranded nucleic acids, and a detection system.
- Nuclease P (1 mg; obtained from Sigma Chemical Company, batch no: 107F0799) was dissolved in 1 ml of water to give a concentration of 22.7 M and stored at 4°C. The activity of this solution was assayed in the following mixture: 0.16 mM NADH, 1 mM ATP, 1 mM PEP, 1 mM MgSO 4 , 20 mM KCI, 0.5 mM adenosine 3 ' ,5 ' -bisphosphate, 1 U pyruvate kinase, 1 U lactate dehydrogenase and 1 U myokinase in 50 mM HEPES buffer, pH 7.2, in a total volume of 1 ml. From the change in absorbance at 340 nm the activity of nuclease P., was solution was found to be 320 U/ml, assuming a molar extinction coefficient of 6220 for NADH.
- Example 2 From the change in absorbance at 340
- a solution of nuclease P, standardised according to Example 1 was serially diluted in 50 mM citrate buffer adjusted to pH 6.5 with NaOH.
- the assay mixture contained 20 mM 3'FADP, 0J mM 4-aminoantipyrine, 2 mM DHSA, 1 g horseradish peroxidase, 0J M glucose and 0J M apoglucose oxidase in a total volume of 0J ml.
- the change in absorbance was monitored at 520 nm in a Dynatech MR7000 plate reader fitted with a thermostatically controlled plate holder set to 25°C.
- Fig. 2 shows the performance of the nuclease P, assay.
- the detection limit (defined as 3 times the standard deviation of the background reading) was 0.2 amol.
- Nuclease S1 was assayed in a similar manner, and the detection limit was 4 amol (Fig. 2).
- Oligonucleotides were synthesised on a CycloneTM DNA synthesiser using the
- the DNA to be labelled with nuclease P was complementary to a region in the middle of the ribonuclease gene containing the K66E mutation.
- This probe was derivatised at the 5' end with a trityl-hexyl thiol group to facilitate linkage to nuclease P
- the sequence was:
- the oligonucleotides were freeze-dried and stored at 4°C until required.
- Nuclease P (5 mg) was dissolved in 0.5 ml 0J M sodium bicarbonate pH 7.5 containing 0J M sodium chloride and desalted by gel filtration on Sephadex G25 (NAP-5 column, Pharmacia) equilibrated with the same buffer. This enzyme solution was incubated with a 50-fold molar excess of 3-(2)-pyridyldithio)-propionic acid N-hydroxysuccinimide ester (SPDP) at room temperature for 30 minutes. Unreacted SPDP was removed by gel filtration on Sephadex G25 (NAP 10 column, Pharmacia) equilibrated with the bicarbonate buffer. The 2-pyridyl disulphide- activated nuclease P 1 was stored at 4°C.
- Nuclease P 1 was linked to 2-pyridyl disulphide as described in Example 4 and stored in 0J M sodium bicarbonate, pH 7.5, containing 0J M sodium chloride at 4°C.
- the K66E or the thiolated S pneumoniae oligonucleotide of Example 3 was dissolved in 0.5 ml 0J M sodium bicarbonate buffer, pH 7.5, containing 0J M sodium chloride to give a final concentration of 0.36 M. This was incubated with activated nuclease P., prepared according to Example 4 at a mole ratio of 1 :2 at room temperature for 45 minutes, followed by an incubation at 4°C for 16 h.
- the conjugate was transferred to 20 mM bis-Tris propane buffer, pH 7.5, containing 1 mM CHAPS by chromatography on Sephadex G25, and purified by ion-exchange chromatography on a Pharmacia Mono Q column. A sodium chloride gradient in the same buffer was used applied to the column and the conjugate was eluted at a molar concentration of 0.25 M.
- Genomic DNA from S pneumoniae was extracted and treated with Pstl to break the DNA up into fragments.
- 95 I of the treated DNA is mixed with 10 I 1 M sodium hydroxide and incubated at room temperature for 10 minutes to denature the DNA before neutralisation with 8 I of 0.5 M sodium citrate buffer, pH 3.0, containing 2.21 M sodium chloride and 0.1% Tween 20.
- 50 I (34 fmol) of the S pneumoniae probe labelled with Nuclease P, described in Example 5 dissolved in 0.1 M Tris-HCI buffer, pH 7.5, containing 7 mM zinc sulphate, 1% (w/v) PVP, 0J % N-lauroylsarkosine and 150 mM sodium chloride, is added.
- the pH is adjusted to about 5.0 by the addition of acetate buffer, and the temperature maintained at 40°C for 10 minutes, after which time more than 95% of unhybridised reporter probe will be hydrolysed.
- the mixture is then introduced into to a commercial immobilised streptavidin column (Pierce ImmunoPure Immobilised Streptavidin) of volume 0.20 mL incubated with TBS.
- the column was eluted with 10 mL of 20 mM Tris-HCI buffer, pH 7.5, containing 7 mM zinc sulphate, 1% (w/v) PVP, 0J % N-lauroylsarkosine and 150 mM sodium chloride.
- the amount of hybrid captured on the column is quantified using the amplification assay described in Example 2: 0J L of the reaction mixture is introduced onto the column, and allowed to produce a coloured product. This is eluted by introducing more of the 20 mM Tris-HCI buffer, pH 7.5, containing 7 mM zinc sulphate, 1% (w/v) PVP, 0J % N-lauroylsarkosine and 150 mM sodium chloride, and its absorbacnce measured.
- This column-based washing and detection step may be conveniently automated using the DuoPrep from Pharmaceutical Technology. This allows the washing and detection solutions to be pumped onto and off the column in a programmed fashion, allowing the coloured product to be recovered in a small volume.
- the method of the present invention can be used to detect hybrids formed between a target nucleic acid and a nucleic acid probe labelled with an enzyme reagent which removes single-stranded nucleic acid.
- This approach eliminates the possibility of cross-talk arising out of the binding of sbm to any single-stranded nucleic acid present.
- the complex formed between hybrid and sbm can be detected using highly sensitive approaches, such as enzyme amplification or chemiluminescence.
- the nucleic acid probe may be labelled with nuclease P, at each end, thereby giving an increase in the overall sensitivity of the detection reaction.
- the method has the additional advantage that it utilises a single probe, which offers cost savings and simplifies the design of assay protocols.
- the nucleic acid probe may be a peptide nucleic acid probe, or another nucleic acid analogue having modified bases or an altered backbone.
- the enzyme reagent may be a protease specific for single stranded peptide nucleic acid.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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CA002356613A CA2356613A1 (en) | 1998-10-12 | 1999-10-12 | Improved hybridisation assay in which excess probe is destroyed |
AU62187/99A AU6218799A (en) | 1998-10-12 | 1999-10-12 | Improved hybridisation assay in which excess probe is destroyed |
EP99949210A EP1121463A1 (en) | 1998-10-12 | 1999-10-12 | Improved hybridisation assay in which excess probe is destroyed |
JP2000576055A JP2002527078A (en) | 1998-10-12 | 1999-10-12 | Improved hybridization assay destroys excess probe |
US09/833,918 US20020090617A1 (en) | 1998-10-12 | 2001-04-13 | Hybridisation assay in which excess probe is destroyed |
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GB9822067.6 | 1998-10-12 | ||
GBGB9822067.6A GB9822067D0 (en) | 1998-10-12 | 1998-10-12 | Column-supported hybridisation assay in which excess probe is destroyed |
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WO2000022165A1 true WO2000022165A1 (en) | 2000-04-20 |
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PCT/GB1999/003383 WO2000022165A1 (en) | 1998-10-12 | 1999-10-12 | Improved hybridisation assay in which excess probe is destroyed |
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EP (1) | EP1121463A1 (en) |
JP (1) | JP2002527078A (en) |
AU (1) | AU6218799A (en) |
CA (1) | CA2356613A1 (en) |
GB (2) | GB9822067D0 (en) |
WO (1) | WO2000022165A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US7252975B2 (en) | 2001-05-09 | 2007-08-07 | The Secretary Of State For Defence | Analytical method and kit |
EP2209905A1 (en) * | 2007-10-17 | 2010-07-28 | 3M Innovative Properties Company | Rapid detection of microorganisms |
US7838236B2 (en) | 2005-08-19 | 2010-11-23 | Enigma Diagnostics Limited | Analytical method and kit |
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GB0029379D0 (en) * | 2000-12-01 | 2001-01-17 | Arrow Therapeutics Ltd | A method for identifying enzyme inhibitors |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0144913A2 (en) * | 1983-12-12 | 1985-06-19 | Miles Inc. | Hybridization assay employing labeled probe and anti-hybrid |
EP0405592A2 (en) * | 1989-06-30 | 1991-01-02 | Sanyo Chemical Industries Ltd. | A method for detection of nucleic acid and reagents for their detection |
EP0780479A2 (en) * | 1995-12-23 | 1997-06-25 | Roche Diagnostics GmbH | Method for quantitative determination of specific nucleic acid sequences |
WO1998019168A1 (en) * | 1996-10-29 | 1998-05-07 | London Biotechnology Limited | Assays and probes with enzyme labels |
GB2324370A (en) * | 1997-04-14 | 1998-10-21 | Stuart Harbron | Method for detecting nucleic acids |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU1020897A (en) * | 1995-11-20 | 1997-06-11 | Trustees Of Boston University | Method and probe for detecting a target nucleic acid sequence |
-
1998
- 1998-10-12 GB GBGB9822067.6A patent/GB9822067D0/en not_active Ceased
-
1999
- 1999-10-12 AU AU62187/99A patent/AU6218799A/en not_active Abandoned
- 1999-10-12 WO PCT/GB1999/003383 patent/WO2000022165A1/en not_active Application Discontinuation
- 1999-10-12 CA CA002356613A patent/CA2356613A1/en not_active Abandoned
- 1999-10-12 EP EP99949210A patent/EP1121463A1/en not_active Withdrawn
- 1999-10-12 JP JP2000576055A patent/JP2002527078A/en not_active Withdrawn
- 1999-10-12 GB GB9924169A patent/GB2346694B/en not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0144913A2 (en) * | 1983-12-12 | 1985-06-19 | Miles Inc. | Hybridization assay employing labeled probe and anti-hybrid |
EP0405592A2 (en) * | 1989-06-30 | 1991-01-02 | Sanyo Chemical Industries Ltd. | A method for detection of nucleic acid and reagents for their detection |
EP0780479A2 (en) * | 1995-12-23 | 1997-06-25 | Roche Diagnostics GmbH | Method for quantitative determination of specific nucleic acid sequences |
WO1998019168A1 (en) * | 1996-10-29 | 1998-05-07 | London Biotechnology Limited | Assays and probes with enzyme labels |
GB2324370A (en) * | 1997-04-14 | 1998-10-21 | Stuart Harbron | Method for detecting nucleic acids |
Non-Patent Citations (4)
Title |
---|
COREY D R ET AL: "STRAND INVASION BY OLIGONUCLEOTIDE-NUCLEASE CONJUGATES", BIOCONJUGATE CHEMISTRY,US,AMERICAN CHEMICAL SOCIETY, WASHINGTON, vol. 6, 1 February 1995 (1995-02-01), pages 93 - 100, XP002024695, ISSN: 1043-1802 * |
FLISS I ET AL.: "Production and characterization of anti-DNA-RNA monoclonal antibodies and their application in Listeria detection", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 59, no. 8, 1993, pages 2698 - 2705, XP000874757 * |
HARBRON S ET AL: "AMPLIFIED ASSAY OF ALKALINE PHOSPHATASE USING FLAVINADENINE DINUCLEOTIDE PHOSPHATE AS SUBSTRATE", ANALYTICAL BIOCHEMISTRY, 1 October 1992 (1992-10-01), XP002072948 * |
STEFFAN R T AND ATLAS R M: "Solution hybridization assay for detecting genetically engineered microorganisms in environmental samples", BIOTECHNIQUES, vol. 8, no. 3, 1990, pages 316 - 318, XP000867831 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7252975B2 (en) | 2001-05-09 | 2007-08-07 | The Secretary Of State For Defence | Analytical method and kit |
US7947476B2 (en) | 2001-05-09 | 2011-05-24 | The Secretary Of State For Defence | Analytical method and kit |
US7838236B2 (en) | 2005-08-19 | 2010-11-23 | Enigma Diagnostics Limited | Analytical method and kit |
EP2209905A1 (en) * | 2007-10-17 | 2010-07-28 | 3M Innovative Properties Company | Rapid detection of microorganisms |
EP2209905A4 (en) * | 2007-10-17 | 2010-10-06 | 3M Innovative Properties Co | Rapid detection of microorganisms |
Also Published As
Publication number | Publication date |
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JP2002527078A (en) | 2002-08-27 |
AU6218799A (en) | 2000-05-01 |
GB9822067D0 (en) | 1998-12-02 |
CA2356613A1 (en) | 2000-04-20 |
GB2346694B (en) | 2001-01-24 |
EP1121463A1 (en) | 2001-08-08 |
GB2346694A (en) | 2000-08-16 |
GB9924169D0 (en) | 1999-12-15 |
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