[go: up one dir, main page]

WO2009026651A1 - Méthode de détection d'un acide nucléique - Google Patents

Méthode de détection d'un acide nucléique Download PDF

Info

Publication number
WO2009026651A1
WO2009026651A1 PCT/AU2008/001284 AU2008001284W WO2009026651A1 WO 2009026651 A1 WO2009026651 A1 WO 2009026651A1 AU 2008001284 W AU2008001284 W AU 2008001284W WO 2009026651 A1 WO2009026651 A1 WO 2009026651A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
nucleic acid
region
single stranded
target nucleic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2008/001284
Other languages
English (en)
Inventor
Aaron Ingham
Moira Menzies
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
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
Priority claimed from AU2007904702A external-priority patent/AU2007904702A0/en
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of WO2009026651A1 publication Critical patent/WO2009026651A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • 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
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention relates to a method for detecting a nucleic acid. More particularly, the invention relates to a method of detecting the nucleic acid and amplifying the resulting signal whereby, in one embodiment, the nucleic acid may be visualised such as in an in situ hybridisation assay or, in another embodiment, wherein the presence or absence of a nucleic acid may be detected such as in an in vitro diagnostic test.
  • nucleic acid amplification is fundamental to a range of biological assays. Application of this method allows for sensitive detection of target nucleic acids that may be present in very low quantities.
  • the field of clinical medicine has benefited greatly from nucleic acid amplification techniques that allow rapid and accurate diagnosis of infectious diseases, genetic disorders and genetic traits.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • SDA self-sustained sequence replication
  • LAMP loop mediated isothermal amplification
  • RCA rolling circle amplification
  • Limiting factors include a requirement for expensive equipment such as thermal cyclers and the associated running costs (electricity and maintenance) , multiple expensive reagents (particularly enzymes and modified nucleotides) , specificity issues, elevated background signal and an incompatibility with other techniques (particularly in situ hybridisation) .
  • the in situ hybridisation technique is used to determine the location of target nucleic acids or proteins in a tissue. Obtaining a discrete signal is therefore vital to interpretation of any in situ result.
  • a labelled probe complementary to the target, is exposed to the tissue. Where present the probe binds to the specific target.
  • the tissue is then washed to remove unbound probe and the location of the bound probe is then visualised by radioactive, chromogenic or fluorescent methods depending on what labels have been incorporated in the probe.
  • the low abundance of many targets in the sample tissue frequently limits the intensity of the signal due to a basic stoichiometry, with 1 probe binding to 1 target.
  • the researcher can use more starting material, attempt to amplify the number of target molecules prior to hybridisation (using any of the previously described methods) , incorporate more signal generating material per probe or amplify the resulting probe signal.
  • the amount of starting material can only be increased occasionally, as the material is often only available in small amounts or in the case of in situ hybridisation, the extra thickness of tissue leads to later difficulties in obtaining clear images for analysis.
  • the streptavidin molecule is conjugated with a signal generating molecule such as horseradish peroxidase (HRP) a 4 -fold increase results.
  • HRP coupled to the probe is not used directly for probe visualisation but rather to activate tyramide derivatives that are themselves coupled to detectable markers such as a fluorescent dye or biotin. Once activated these derivatives then bind nucleic acids in the localised area. As each incorporated HRP molecule can activate many tyramides the signal is amplified accordingly. Overall, these processes are expensive, complex, involve numerous reagents and have limited direct amplification potential (still 1 probe for 1 target) . Additionally, because the tyramide system relies on the deposition of the activated dinitrophenyl adjacent to the site of hybridisation the resulting signal may be diffuse and non-specific.
  • An alternative method for in situ hybridisation signal amplification involves branched DNA.
  • This method uses numerous non-isotopic labelled oligonucleotide probes to generate and amplify the signal.
  • the method is complex and expensive as it can involve more than 100 oligonucleotides, each of which must be labelled, for the detection of each mRNA.
  • NAA non- enzymatic amplification
  • the process of WO 2003/046512 involves introducing a reference primer that is covalently coupled to a probe that binds to a target molecule.
  • the probe may be an antibody or the like but may also be a nucleic acid. Only the probe is specific to the target; the reference primer is a nucleic acid sequence unrelated to the sequence of the probe or the target.
  • two amplification primers, designated amplifier I and amplifier II are added to amplify the signal.
  • amplifier primer I is a symmetrical molecule with its 3' half sequence fully complementary to the reference primer, and its 5' half sequence fully complementary to the 5' half of the amplifier II primer.
  • the amplifier II primer is also a symmetrical molecule, and its 5' half sequence is complementary to the 5' half of amplifier I and its 3' half is complementary to the 3' half of amplifier I.
  • the gene specific probe, reference primer and/or amplifier I and amplifier II may contain detectable labels, for example, they may be biotinylated or radioactively labelled to allow detection.
  • the NEA method requires a first hybridisation step during which time the gene specific probe binds to the complementary target sequence, if present.
  • the covalently attached reference primer being unrelated in sequence to the probe or target, does not and is thus left single stranded and extending from the probe target complex.
  • a second signal amplification step is then conducted that requires the addition of the two amplifier sequences.
  • the amplification process initiates with the complementary regions of amplifier I and the overhanging reference primer hybridising, leaving the unrelated region of amplifier I overhanging. This region is however complementary to part of amplifier II allowing these to hybridise but leaving an overhanging single stranded section of amplifier II. This may then bind to the complementary region of another amplifier I with the remaining sequence left overhanging.
  • the continued hybridisation of amplifier I to amplifier II then repeats through many cycles. Since each one of the many amplifier I and amplifier II molecules now stacked in the complex is labelled an intense signal is generated after a number of cycles.
  • the initial binding depends upon the probe only and the amplification cascade that takes place is independent of the correctness or otherwise of this binding or correct linkage of the reference primer to the probe.
  • the sequence of the reference primer and amplifiers must be unique from any present in the sample otherwise there is a possibility of false positives leading to incorrect and inappropriate images.
  • difficulty may be experienced in linking the reference primer to the probe and in subsequent hybridising of amplifiers to the reference primer due to the small size of amplifiers and steric interference in the system.
  • a further issue stemming from the small size of these probes is that this limits the amount of probe that may be incorporated and the researcher is restricted to synthetic production of the probes.
  • a method of improving the sensitivity of hybridisation probes involving random cleavage and ligation of sequence derived from a target nucleic acid is described in US Publication No. 20010051342.
  • a labelled probe designed to anneal to a target strand is fragmented, for example by the action of a restriction endonuclease, and the fragments are ligated using a DNA ligase to produce a library of DNA molecules each composed of a permuted combination of fragments from the digest.
  • the library itself is used in an in situ hybridisation method to form a network of probe molecules, or individual, permuted probes can be isolated from the library and amplified for use to increase assay reproducibility and provide control over the extent of network formation. Nevertheless control is not absolute as the permuted probe is produced randomly and correctly detecting, isolating and amplifying a desired probe from among the many in the library first produced appears difficult to achieve with certainty in a cost effective manner.
  • the present invention provides a method of amplifying a signal to detect a target nucleic acid, comprising the steps of:
  • a first single stranded nucleic acid probe comprising a first region substantially complementary to sequence in a segment of said target nucleic acid, a third region containing sequence substantially identical to that in said first region and in the same orientation and, optionally, a second region spacing said first region from said third region, and a second single stranded nucleic acid probe substantially complementary to the first single stranded nucleic acid probe, the first probe and/or the second probe being labelled with a detectable label; (2) contacting a sample which putatively contains the target nucleic acid with the first and second strands and thereafter maintaining conditions appropriate for hybridisation; whereby amplification of a signal is achievable following binding of the first probe to the target nucleic acid through immobilisation of a plurality of detectable labels by way of repeated, alternate binding of the second probe to the first probe and the first probe to the second probe.
  • first and the second single stranded probes are provided by denaturing a double stranded probe comprising the first and second strands.
  • the target nucleic acid is immobilised to a solid support.
  • the target nucleic acid hybridises with a capture oligonucleotide bound to the solid support.
  • the capture oligonucleotide is covalently bound to the solid support.
  • a plurality of target nucleic acids are immobilised such as by hybridisation to an array of capture oligonucleotides.
  • the target nucleic acid is immobilised to a dipstick and amplification of the signal provides a yes/no indication of the presence of the target nucleic acid.
  • the target nucleic acid is localised within a biological sample.
  • the biological sample is a tissue or cell preparation, for example a tissue section or fluid containing cells applicable to a slide.
  • a first single stranded nucleic acid probe comprising a first region substantially complementary to a sequence in a segment of said target nucleic acid, a third region containing sequence substantially identical to that in said first region and in the same orientation and, optionally, a second region spacing said first region from said third region, and a second single stranded nucleic acid probe substantially complementary to the first single stranded probe, the first probe and/or the second probe being labelled with a detectable label; (2) contacting a biological sample in which the target nucleic acid is localised with the first and second probe and thereafter maintaining conditions appropriate for hybridisation; and
  • the first single stranded probe and the second single stranded probe are provided by denaturing a double stranded probe comprising the first and second strands.
  • the methods of the invention do not rely upon covalent linkage of the probe to a reference primer nor multiple hybridisation steps; but involves direct binding of a target sequence with a labelled probe through the first region on the first strand and subsequent signal amplification in a single step. While not wishing to be bound by theory, it is believed that amplification of the signal is achieved through repeated hybridisation of complementary regions of the respective first and second strands.
  • the first strand binds the target nucleic acid through the first region, which leaves the second and third regions overhanging. These may in turn bind complementary sequence on the second strand. This leaves a region of the second strand overhanging, to which a complementary region of another first strand binds, and so on as illustrated in Fig. 2 to introduce multiple detectable labels to the "stack" all of which are immobilised to and therefore localised in the immediate vicinity of the target nucleic acid.
  • the present invention provides a method of forming a nucleic acid complex, comprising the steps of:
  • first single stranded nucleic acid probe comprising a first and third region containing substantially identical sequence and in the same orientation and, optionally, a second region spacing said first region from said third region, and a second single stranded nucleic acid probe substantially complementary to the first single stranded probe;
  • the first single stranded nucleic acid probe and the second single stranded nucleic acid probe are provided by denaturing a double stranded probe comprising the first and second strands.
  • the invention provides a nucleic acid complex comprising (a) a first single stranded nucleic acid probe comprising a first and third region containing substantially identical sequence and in the same orientation and, optionally, a second region spacing said first region from said third region (b) a second single stranded nucleic acid probe a second single stranded nucleic acid probe substantially complementary to the first single stranded probe (c) further additions of the first single stranded nucleic acid probe and the second single stranded nucleic acid probe; whereby complex formation takes place through alternate binding of the first probe to the second probe and the second probe to the first probe.
  • a target nucleic acid is present, for example to locate or anchor the nucleic acid complex.
  • the first region of the first single stranded nucleic acid probe is substantially complementary to sequence in a segment of said target nucleic acid.
  • the invention provides a double stranded nucleic acid probe for amplifying a signal to detect a target nucleic acid, comprising a first strand comprising a first region substantially complementary to sequence in a segment of said target nucleic acid, a third region containing sequence substantially identical to that in said first region and in the same orientation and, optionally, a second region spacing said first region from said third region, and a second strand comprising a sequence substantially complementary to the first strand, wherein at least one strand is labelled with a detectable label.
  • the invention provides a composition for amplifying a signal to detect a target, comprising a first single stranded nucleic acid probe comprising a first region substantially complementary to sequence in a segment of said target nucleic acid, a third region containing sequence substantially identical to that in said first region and in the same orientation and, optionally, a second region spacing said first region from said third region, and a second single stranded nucleic acid probe comprising a sequence substantially complementary to the first strand, wherein at least one nucleic acid probe is labelled with a detectable label.
  • amplification of a signal from the detectable label is achieved following denaturation of a double stranded probe through binding of the first strand to the target nucleic acid and then through repeated alternate binding of the second strand to the first strand and the first strand to the second strand.
  • Figure 1 is a schematic diagram illustrating construction of probe for use in the method of the invention using recombinant techniques .
  • Figure 2 is a schematic diagram illustrating the principle of signal amplification according to the invention.
  • the "explosion" icon indicates a detectable label .
  • Figure 3 shows photomicrographs giving comparative in situ hybridisation results using the optimised probe described here versus conventional methods for the Muc5 gene.
  • a positive result is indicated by a dark staining.
  • the background is stained red for contrast.
  • a section treated with No probe shows background light staining only (top left corner) .
  • a section probed with the spacer shows background light staining only (top right corner) .
  • the conventional probe produces the expression pattern seen in bottom right hand corner while our repeat probe produces a similar but much more intense pattern in the bottom left hand corner.
  • the visualisation reaction was left for 12 hours.
  • a comparable result could be obtained using the optimised probe in 2 hours.
  • Figure 4 shows photomicrographs which compare of in situ result generated from conventional probe to result generated from optimised probe developed using technique described here for two further genes, XDH and NOS2A.
  • Figure 5 shows photomicrographs confirming that an optimised probe of the invention detects signal of FGFlO and BMP4 in ovine foetal skin.
  • nucleic acid refers to deoxyribonucleic acid and ribonucleic acid and the like in all their forms.
  • the nucleic acid may comprise natural or non-natural nucleic acid, or combinations thereof.
  • the nucleic acid can comprise a nucleic acid analog or chimera comprising nucleic acid and nucleic acid analog monomer units, such as 2- aminoethylglycine, phosphorothiocite DNA, 2'-O-methyl RNA (OMe), 2 / -0-methoxy-ethyl KNA (MOE), N3'-P5' Phosphoroamidate (NP), 2' -fluoro-arabino nucleic acid (FANA) , Morpholino posphoroamidate (MF) , cyclohexene nucleic acid (CeNA) , Tricyclo-DNA (+c DNA) , peptide nucleic acid (PNA) , or locked nucleic acid (LNA) .
  • nucleic acid analog or chimera comprising nucleic acid and nucleic acid analog monomer units, such as 2- aminoethylglycine, phosphorothiocite DNA, 2'-O-methyl RNA (OMe),
  • nucleic acid analogues are used which enhance hybridisation between complementary sequences.
  • locked nucleic acids represent a class of conformationally restricted nucleotide analogues described, for example, in WO 99/14226 which hybridise more strongly to both DNA and RNA than naturally occurring nucleotides.
  • Introduction of a locked nucleotide into a nucleic acid improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
  • LNAs known in the art, for example, those disclosed in WO 99/14226 and in Latorra D, et al., 2003. Hum. Mutat. 22: 79-85. More specific binding can be obtained and more stringent washing conditions can be employed using LNA analogs, with the advantage that the amount of background noise is reduced significantly.
  • the method comprises providing a first single stranded nucleic acid probe comprising a first, second and third region.
  • the first region is substantially complementary to sequence in a segment of the target nucleic acid.
  • a nucleic acid sequence is "substantially complementary" to another nucleic acid sequence if the two sequences are capable of hybridising.
  • a nucleic acid sequence is substantially complementary to another nucleic acid sequence if greater than 85% of the sequence 1 "3
  • stringent conditions for hybridization or annealing of nucleic acid molecules are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 5O 0 C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Fi ⁇ oll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 75OmM NaCl, 75mM sodium citrate at 42°C.
  • SDS sodium dodecyl sulfate
  • formamide for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Fi ⁇ oll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 75OmM NaCl, 75mM sodium citrate at 42°C.
  • the third region of the probe contains sequence substantially identical to the first region and in the same orientation.
  • a nucleic acid sequence is substantially identical to another nucleic acid sequence if the nucleic acid sequences are at least 85% identical, more typically 90% identical, still more typically 95% identical, even more typically 99% identical, yet more typically 100% identical.
  • the first and third regions of the probe are identical in their sequence and orientation.
  • first and third regions of the nucleic acid strands are complementary to the target nucleic acid.
  • the second region spaces the first region from the third region.
  • the second region may be any sequence provided it does not adversely affect detection of the target nucleic acid.
  • the second region comprises non-specific and unique nucleic acid sequence and separates the first and third region. It will be appreciated that this spacer sequence does not bind nucleic acid in the sample but as part of the amplification process the probes may hybridise through this region as well as through the first and third regions.
  • the nucleic acid strands may include additional non-specific and unique sequence at one or both ends provided that it does not interfere with primary binding to target or subsequent hybridisation of the first and third regions .
  • the second single stranded nucleic acid probe comprises a region that is substantially complementary to the second region of the first single stranded nucleic acid probe.
  • the second single stranded nucleic acid probe comprises a region that is complementary to the second region of the first single stranded nucleic acid probe, and therefore hybridisation through the respective second regions of the probes is possible in addition to hybridisation through the first and third regions of the respective probes .
  • the probes are substantially complementary along their length and capable of hybridising throughout the length of the overhang.
  • the method comprises providing a first single stranded nucleic acid probe and a second single stranded nucleic acid probe.
  • the first and second single stranded nucleic acid probes are DNA strands.
  • the first and second single stranded nucleic acid probes are RNA strands.
  • the first single stranded nucleic probe is a DNA strand and the second single stranded nucleic acid probe is an RNA strand.
  • the first single stranded nucleic acid probe is an RNA strand and the second single stranded nucleic acid probe is a DNA strand.
  • first and second single stranded nucleic acid probes are provided by denaturing a double stranded probe comprising the first and second strand.
  • the method comprises providing a double stranded probe with a first strand and a second strand.
  • the probe is a double stranded DNA probe.
  • the probe is a double stranded RNA probe.
  • the probe comprises a DNA strand and a complementary RNA strand.
  • the probe may comprise two single but complementary strands of DNA or RNA that may be added together or sequentially and are constructed as described above.
  • nucleic acids appropriate for use in the present invention is well within the capability of the person skilled in the art.
  • the completed nucleic acid strands or probes will contain 300-600 nucleotides, with each of the three sections ranging from 100-200 nucleotides in length.
  • a probe for use in the method of the invention may be prepared using recombinant DNA technology. By way of example, the following is a general scheme, as illustrated in Figure 1, which may be employed to prepare a probe for use in the method of the invention: Amplification of Region 1
  • Step 1 The target nucleic acid sequence is identified and a 100-200 nucleotide region is amplified using PCR which is complementary to the target nucleic acid.
  • the 5' region of the 3' primer used in this amplification is typically complementary to the 5' region of the fragment corresponding to the second region.
  • the 5' region of the 5' primer used in this amplification typically contains a T7 sequence separated from the target sequence by a novel restriction site.
  • Step 2 A non-specific and unique DNA sequence of 100-200 nucleotides may be amplified using PCR or generated as a synthetic fragment.
  • the 3' primer typically includes a second unique restriction enzyme site to facilitate linkage of the third region.
  • Regions 1 and 2 Joining of Regions 1 and 2 Step 3.
  • the target nucleic acid sequence and nonspecific sequence are mixed and denatured at 94°C.
  • the solution is cooled to 5O 0 C to allow annealing. In some cases the target sequence will anneal to the nonspecific spacer fragment.
  • Step 4. The single stranded overhangs can then be filled using Taq polymerase to generate a construct containing region 1 and region 2.
  • Preparation of Region 3 Step 5 Amplification of probe region 3.
  • the target nucleic acid sequence identical to that produced in step 1 is amplified using PCR.
  • the 5' region of the 3' primer used in this amplification should be complementary to SP6 and separated from the target sequence by the same restriction site incorporated in step 1.
  • the 5' primer typically includes the second unique restriction enzyme site. While the 5' region of the 5' primer used in this amplification may- contain the 3' region of the non-specific spacer.
  • step 6 Joining Region 3 to Regions 1 and 2 Step 6.
  • the products of step 4 and step 5 are digested with the second restriction enzyme and ligated.
  • the resulting product will be the desired probe containing regions 1, 2 and 3.
  • Step 7 The desired probe can then be amplified in a PCR using SP6 and T7. DIG labelled nucleotides can be incorporated at this stage.
  • Step 8 The T7 and SP6 sites may be removed by digesting with the first restriction enzyme however, this is not vital as the method will work even if these additional sequences are left on the probe complex.
  • Step 9 The probe is ready for use in the method of the invention.
  • the probe can be prepared by synthetic methods .
  • the probe may be prepared synthetically by producing one continuous strand and its complement or designing a series of complementary and overlapping oligonucleotides that can be renatured and annealed to form the target probe.
  • Nucleic acid analogs such as those described above may be incorporated into the nucleic acid probe during synthesis.
  • At least one strand comprises a suitable label allowing detection.
  • a probe may be labelled directly and used as a DNA probe.
  • transcription may be initiated from a suitable promoter such as an SP6 and / or T7 site to generate a dsRNA probe, a ssRNA or a hybrid of DNA and RNA.
  • Labels according to the invention may be applied by methods well understood by the person skilled in the art. Appropriate methods for labelling include PCR, nick translation, end labelling, intercalation, in vitro transcription and random primers. Such methods are described in, for example, Sambrook, J., E. F. Fitch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed.
  • Standard reagents may be used with any of these methods including digoxigenin (DIG) , biotin, horseradish peroxidase, chromophores such as diaminobenzidine (DAB) , NBT and BCIP, fluorophores such as FITC, Cy3, Cy5 and derivatives and radioactive labels including 3H, 32P, 33P, 35S.
  • DIG digoxigenin
  • biotin horseradish peroxidase
  • chromophores such as diaminobenzidine (DAB) , NBT and BCIP
  • fluorophores such as FITC, Cy3, Cy5 and derivatives
  • radioactive labels including 3H, 32P, 33P, 35S.
  • the label is incorporated into the length of probe as described in the PCR method above. End labelling might also be suitable although a much lower amount of label will be incorporated.
  • the labels may be detected in a manner well understood by the person skilled in the art.
  • a biotinylated nucleic acid may be detected by use of the very specific binding of biotin to avidin or streptavidin, which are conjugated with enzyme systems which allow signal detection.
  • DIG may be detected through binding of a similar anti-DIG antibody-enzyme conjugate.
  • colour development occurs through the action of the enzyme on a chromogen such as NBT/BCIP, which results in deposition of a blue / brown precipitate at the site of hybridisation.
  • the system may be enhanced in conventional systems by using a tyramide signal amplification system. However, it will be appreciated by persons skilled in the art that additional signal amplification is not necessary in the present invention.
  • the probes are lengthy molecules with a considerable number of labels applied thereto. Accordingly the effect of stacking of the strands in amplifying the signal increases rapidly with each cycle.
  • detecting a target nucleic acid involves visualising a nucleic acid which is known or expected to be present in the sample.
  • the target nucleic acid is a mRNA and, in particular, a mRNA of relatively low abundance or one which is localised in a sample.
  • the target nucleic acid is a DNA molecule.
  • the method of the invention can be applied to the detection of a target nucleic acid in any applications in which a conventional probe can be used to detect a target nucleic acid.
  • the method of the invention is used for in situ hybridisation.
  • the probe is designed to bind a mRNA for a selected gene localised in a portion of a sample or genomic DNA. In this way the distribution of the expression product may be determined.
  • the method of the invention is used for Southern, Northern or Dot blot hybridisation.
  • the probe is designed to bind to target DNA or RNA immobilised on a membrane support.
  • Southern hybridisation, Northern hybridisation and dot blot hybridisation are described in, for example, Sambrook, J., E. F. Fitch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
  • the method is used for detecting a nucleic acid which may or may not be present in a sample such as in a diagnostic test.
  • the method could be used in detection of viral, bacterial, protozoal, parasitic, fungal, animal or plant nucleic acids.
  • the method may also be used to detect sequences, either mRNA or genomic DNA, that are indicative of a disease state such as in cancer or even in genetic disorders.
  • the method is used for detecting polymorphisms or specific alleles.
  • a target nucleic acid may be localised in a biological sample such as a tissue section or similar, in particular, when placed on a slide.
  • a biological sample such as a tissue section or similar
  • photomicrographs such as in Figures 3 to 5 may be produced demonstrating the location of the target nucleic acid.
  • the target nucleic acid may be present in solution in a sample presented for analysis.
  • the target nucleic acid may be immobilised to a solid support.
  • solid support envisages any solid phase material irrespective of scale provided that a nucleic acid, once secured to the solid support, may be removed or separated in some fashion from the solution.
  • the solid support may be the side of a vessel or a well within a vessel, a three-dimensional matrix through which the solution passes such as a cartridge or filter, a bead, a capsule, a microparticle or any other material which may be ultimately be separated from the solution.
  • the target nucleic acid is sequestered to a solid support by way of hybridisation to an oligonucleotide which is itself immobilised to the solid support.
  • This capture oligonucleotide would advantageously bind to a different region of the target nucleic acid from that to which the first single stranded nucleic acid probe binds.
  • Advantageously binding to the oligonucleotide would orientate the nucleic acid so that the region to which the first single stranded nucleic acid probe binds is exposed.
  • the means of fixing the capture oligonucleotide may be any suitable means.
  • the capture probe is chemically modified to allow covalent linkage to the solid support.
  • antibodies, avidin or streptavidin to be immobilised onto the surface of the solid phase can be attached by either physical adsorption or through covalent linkage.
  • Various chemistries are available. The most common are introduction of an amine, carbonyl, carboxyl or thiol group to the capture oligonucleotide.
  • the functional group introduced is chosen so as to be capable of reaction with a functional group present on the solid support.
  • the linkage of the capture oligonucleotide takes place through reaction of the introduced group with a functional group on the solid support.
  • the reaction may be formation of a Schiff base where an aldehyde group reacts with an amine followed by reduction, esterification or amide formation, but complex chemistries may also be employed. If a covalent linkage is employed a spacer of the correct size can be introduced to ensure that the immobilised capture probe can move freely to hybridise with the target nucleic acid. It will be appreciated that covalent linking techniques may be employed to link a plurality of capture oligonucleotides, for example to create a micro- or macroarray. A vast number of different linking chemistries can be used, depending on the immobilisation substrate, provided there is a suitable active group on the membrane surface.
  • N-oxysuccinimide esters are covalently linked to the surface of the plate, typically by a spacer which is linked covalently to the surface of the plate at one end and to the N-oxysuccinimide moiety at the other.
  • DNA with primary amines added synthetically or by in vitro manipulation can be directly coupled to the reactive N-oxysuccinimide esters to attach a capture oligonucleotide to a solid support .
  • amine a primary amine onto a nucleic acid.
  • the most common method is to incorporate the amine onto either the 5 1 or 3 1 end of the molecule during synthesis.
  • the amine is attached to the phosphoribose backbone via a carbon linker of either 3, 6 or 12 carbons so as to extend the oligonucleotide away from the plate surface, thus allowing greater access and enhancing hybridisation.
  • hapten linkage such as biotin- avidin or biotin-streptavidin systems may be employed.
  • an oligonucleotide probe which is biotinylated may bind to avidin or streptavidin fixed to the solid support by chemical means.
  • the target nucleic acid may be immobilised by binding to an antibody bound to a solid support or an antibody to which is bound an oligonucleotide probe to which the target nucleic acid hybridises.
  • Antibody-dependent capture usually employs an antibody capture line deployed on the solid support and an oligonucleotide probe of complementary sequence.
  • oligonucleotide capture probes may be immobilised directly onto a membrane. UV irradiation is one way to ensure covalent bonding of such a probe to a nylon membrane. Alternatively this may be achieved by passive adsorption of a BSA-labelled oligonucleotide probe or an unlabelled oligonucleotide probe to the surface.
  • a plurality of capture oligonucleotides are fixed to the surface of a solid support.
  • Each capture oligonucleotide will capture a specific target nucleic acid and, once captured, each captured target nucleic acid may be visualised using the method of the present invention. Accordingly, with appropriate design of capture oligonucleotides and nucleic acid probes a microarray or macroarray may be prepared and a methodology for its use developed to obtain information about a plurality of target nucleic acids in each analysis.
  • a target nucleic acid may be the subject of a simple "on/off test" such as when immobilised to a dipstick.
  • a colour change can be detected through the methods of the invention if binding of the target nucleic acid to the capture oligonucleotide (s) immobilised to the dipstick takes place.
  • the capture oligonucleotide (s) may be immobilised across a large portion of the dipstick or in a specific area of the dipstick so as to create a distinctive symbol or message, for example by completing a line, so that a positive test may be easily visualised.
  • a nucleic acid lateral flow detector may be employed.
  • nanoparticles coated with a capture oligonucleotide bind the target nucleic acid. This nanoparticle complex flows laterally through a series of overlapping membranes and is captured on a capture line where the signal may be visualised by the method of the invention.
  • nucleic acids there may be non-specific binding of target nucleic acids to a solid support.
  • physical adsorption of nucleic acids to a solid surface may take place.
  • Nucleic acids can be immobilised onto nitrocellulose membranes by simply air-drying or baking the membrane. Air-drying typically involves exposure for 2 to 8 hours. The alternative is oven-drying at 8O 0 C for 2 hours.
  • Physical adsorption may be enhanced by modification to the nucleic acid molecule which, it will be appreciated, may assist in fixing a capture oligonucleotide by this method.
  • a poly-T tail may be added to the capture oligonucleotide to enhance adsorption and to increase the prospects (since the poly-T tail binds more strongly) that the probe is correctly orientated for hybridisation.
  • Techniques for immobilisation of nucleic acids are described, for example, by Jones, KD "Membrane Immobilisation of Nucleic Acids, Part 2: Probe Attachment Techniques", IVD Technology 2, No. 3 (2001) page 59, the contents of which are incorporated herein by reference.
  • region 1 for use in constructing a probe for ovine versions of the genes MUC5, XDH, NOS2A, FGFlO and BMP4, sequence was amplified from a cDNA molecule for each of the genes using PCR (step 1 of Figure 1) .
  • the primer pairs that were used to amplify each gene specific region are listed in Table 1.
  • NOS2A NOS2A-F ⁇ '-TCAGAGCCACGATCCTCTTT-S' 250bp
  • BMP4 -R 5 ' -ATACGATGAAAGCCCTGCTC-3 ' These primers were used in a PCR consisting of 30 cycles of: 94 0 C for 30 seconds, 55 0 C for 30 seconds and 72 0 C for 30 seconds.
  • Reactions were performed in 20 ⁇ l total volume and contained 10 ng of either ovine gut or ovine foetal skin cDNA, 0.9 ⁇ M of specific primers and 1 Unit of Taq DNA polymerase in 1 x PCR buffer [45mM Tris.HCL (pH8.8); HmM (NH 4 J 2 SO 4 ; 4.SmM MgCl 2 ; 6.7mM 2- mercaptoethanol; 4.4 ⁇ M EDTA (pH8.0); ImM each dNTPs] .
  • the PCR products were then agarose gel purified and cloned into a commercial PCR cloning vector (pCR2.1; Invitrogen) to use as a template for the generation of region 1.
  • PCR was performed in a total of 50 ⁇ l containing; 5ng of plasmid (containing gene specific region, generated above); 2.5 Units of Taq DNA polymerase; 1 x PCR buffer and 0.9 ⁇ M of each gene specific primer pair listed in Table 2.
  • PCR conditions were as follows, 30 cycles of: 94°C for 30 seconds, 55°C for 30 seconds and 72 0 C for 30 seconds.
  • Muc5-AR 5' -AGACAGGGGGCAGAGCGT GACCAGGCCGTCCAGCTT-3 '
  • Region 2 was generated by amplifying a 187bp intron sequence from ovine genomic DNA using the following primers; 5 ' -ACGCTCTGCCCCCTGTCT-3 ' and 5' -
  • CTGAATTCTGCAGGGAGAGG-S ' Reactions were performed in 20 ⁇ l total volume and contained 200 ng of ovine genomic DNA, 0.9 ⁇ M of specific primers and 1 Unit of Taq DNA polymerase in 1 x PCR buffer. PCR conditions involved 30 cycles of: 94 0 C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds. (Step 2 of Figure 1).
  • the amplified nucleic acid for regions 1 and 2 for each gene were subsequently gel purified and eluted in 50 ⁇ l of 1OmM Tris buffer.
  • 0.5 ⁇ l of DNA of amplified region 1 and 2 were mixed in 1 x PCR buffer with 2.5 Units of Taq DNA polymerase in a total volume of 50 ⁇ l and incubated at 95°C for 5 minutes to denature the nucleic acid molecules.
  • the solution was subsequently cooled to 5O 0 C for 30 seconds to allow annealing of region 1 to region 2 and incubated at 72 0 C for 15 minutes to extend the product, (see step 3 of Figure 1) .
  • T7 5'primer eg T7-Muc5; see Table 2
  • 3' primer used to amplify region 2 5'-CTGAATTCTGCAGGGAGAGG-S'
  • PCR conditions involved 30 cycles of: 94 0 C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds.
  • region 3 of the probe sequence was amplified from the same plasmid as for Region 1 using the primer pairs listed in Table 3 (Step 5 of Figure 1) .
  • the PCR was performed in a total of 50 ⁇ l containing; 5ng of plasmid (containing gene specific region, generated above); 2.5 Units of Taq DNA polymerase; 1 x PCR buffer and 0.9 ⁇ M of each gene specific primer pair listed in Table 2.
  • PCR conditions were as follows, 30 cycles of: 94 0 C for 30 seconds, 55°C for 30 seconds and 72 0 C for 30 seconds.
  • the amplified product was gel purified.
  • Table 3 Primer pairs for generation of Region 3 Gene Primer Primer sequence Size of amplified fragment
  • Regions 1 and 2 and Region 3 were digested with EcoRl and subsequently ligated (see Step 6 of Figure 1) with T4 DNA ligase.
  • the ligated DNA was gel purified and cloned into a commercial PCR cloning vector (pCR2.1; Invitrogen) to use as a template for the generation of labelled probes. This also allows the construct to be verified by- sequencing. DNA probes are generated using ROCHE PCR DIG probe synthesis kit.
  • the probe is labelled with DIG-dUTP in a PCR using primers complementary to the T7 and SP6 sequences at the respective ends of the ligated products (see Step 7 of Figure 1).
  • the T7 primer used was S'-TAATACGACTCACTATAGGG-S' and the SP6 primer used was 5'- ATTTAGGTGACACTATAGA-S '. If the researcher wants to remove the T7 and SP6 priming sites from the probe, then the resulting amplified product can be digested with the appropriate restriction endonuclease (Sad) to remove the T7 and SP6 priming sites (Step 8 of Figure 1) .
  • Sad restriction endonuclease
  • Probe labelling DNA probes generated in Example 1 were labelled with Digoxigenin (DIG) -dUTP using the ROCHE PCR DIG Probe Synthesis Kit. The labelling reaction was carried out as instructed by the manufacturer.
  • DIG Digoxigenin
  • Tissue sections from ovine gut and ovine foetal skin were cut at 4 and 5 micron thickness, repectively and placed on Superfrost plus slides. The slides were then heated to 65 0 C for 15 minutes. The slides were subsequently rehydrated by washing the slide as follows:
  • the optimal proteinase K concentration was determined for each tissue type by setting up a series of digestion conditions with varying concentrations of proteinase K. The tissues were subsequently stabilised by rinsing in
  • the slides were washed in SSC at increasing stringency. Specifically, the slides were washed as follows:
  • the slides were washed in buffer I (ROCHE PCR DIG Probe Synthesis Kit) for 5 minutes at room temperature. Each slide was then incubated with 500 ⁇ l blocking buffer II [0.5% blocking reagent (ROCHE) in buffer I] for 30 minutes at 38 0 C in a humid chamber, making sure that the buffer II was at room temperature when used. The blocking buffer was subsequently removed and the slide treated with 500 ⁇ l of anti-DIG antibody conjugate (1 in 2000) in blocking buffer II for 30 minutes at 38 0 C in a humid chamber. The anti-DIG antibody conjugate was then removed and the slide washed in buffer I at room temperature for 2 x 10 minutes.
  • buffer I ROCHE PCR DIG Probe Synthesis Kit
  • the slides were subsequently washed in buffer III [10OmM Tris-HCl; 10OmM NaCl; pH 9.5] at room temperature for 5 minutes.
  • 500 ⁇ l of colour development solution [0.5mg/ml NBT; 0.1875mg/ml BCIP; 5mM levamisole; 5OmM MgCl 2 ; in buffer III] was then added to each slide and the slide was allowed to develop in a humid chamber in the dark at room temperature (reaction can take 2 hours - overnight) .
  • the reaction was stopped by washing slides in buffer IV [ImM Tris-HCl; 0. ImM EDTA; pH ⁇ .O] for 2 x 15 minutes at room temperature.
  • Counter-staining was performed by incubating the slides in Nuclear Fast Red for 5 minutes, followed by a rinse in H 2 O, and dehydration of the slides through increasing concentrations of ethanol. The slides were cleared in xylene, and mounted with DEPEX.
  • the mounted slides were examined and photographed under light microscopy.
  • Example 3 In situ hybridisation of tissue with the MUC5 gene sequence.
  • Example 4 In situ hybridisation of ovine gut sections with XDH and NOS2A gene sequence.
  • in situ hybridisation was carried out as described in Example 2 above using the full length probe or a probe prepared in line with conventional methodology to contain only a single region capable of hybridising corresponding to the first region to identify XDH or NOS2A expression in ovine gut sections.
  • the results of the hybridisation are shown in Figure 4.
  • Example 5 In situ hybridisation of ovine foetal skin with FGFlO or BMP4 gene sequence using full length probes containing Regions 1 to 3. To determine whether the use of a probe according to the invention as described herein is effective for other tissue types, in situ hybridisation was carried out as described in Example 2 above using the full length probe for genes likely to be expressed in these tissues. Ovine foetal skin was hybridised as described in Example 2 with full probe to FGFlO or BMP4, or with no probe. The results are shown in Figure 5.
  • the full length probe was able to detect signal of FGFlO and BMP4 in ovine foetal skin.
  • Example 6 Detection of nucleic acid in solution.
  • DNA-BIND ® Corning Costar
  • lOO ⁇ l of a 5' amine modified oligonucleotide was added to each well in Oligo Binding Buffer (50 mM Na2PO4, pH 8.5; I mM EDTA) at a concentration of 25pmol/well or greater. The mixture was incubated overnight at 4 0 C or for 1 hour at 37 0 C. Uncoupled oligonucleotide was removed by washing the plate three times with maleate buffer (10OmM maleate, 150 mM NaCl, pH 7.5). The unreacted DNA-BIND active groups were blocked by adding 200 ⁇ l of 3% BSA in Oligo Binding Buffer and the plate incubated for 30 minutes at 37 0 C.
  • Oligo Binding Buffer 50 mM Na2PO4, pH 8.5; I mM EDTA
  • the process involved hybridising in a solution of 5xSSC; 0.05% SDS; 0.005% BSA (RNase-free BSA) and for DNA, the hybridisation solution was 5xSSC; 0.1% SDS.
  • Double stranded DNA must be denatured prior to hybridising it to the capture oligonucleotide to ensure adequate hybridisation efficiency. The DNA was denatured by heating to 95 0 C for 6 minutes then quickly transferring the tube to ice for 1 minute.
  • hybridisation For hybridisation, lOO ⁇ l/well of hybridisation solution containing the target nucleic acid was added and the plate was incubated for 60 minutes at a temperature that was 5 0 C (or lower) below the temperature of dissociation for the capture oligonucleotide. After hybridisation wells were washed with preheated 2xSSC; 0.1% SDS twice and soaked for 5 minutes. The temperature of this solution was the same as the hybridisation temperature. A DIG-labelled DNA detection probe, which is homologous to the 5' region of the target nucleic acid, was then used to amplify the signal. The probe was generated as described in Example 1 and labelled with digoxigenin (DIG) -dUTP using the ROCHE PCR DIG Probe synthesis kit. The Labelling reaction was carried out as instructed by the manufacturer. The probe was denatured on a heating block at 95 0 C for 6 minutes, then placed on ice for 1 minute. The probe was then added to the hybridisation solution and lOO ⁇ l added to each well.

Landscapes

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

Abstract

L'invention porte sur une méthode d'amplification d'un signal en vue de la détection d'un acide nucléique cible consistant: (a) à obtenir une première sonde d'acide nucléique simple brin comprenant: une première région complémentaire d'une partie de la cible, une troisième région sensiblement identique à la première (dans la même orientation), et (facultativement) une deuxième région d'écartement située entre la première et la troisième ainsi qu'une deuxième sonde d'acide nucléique simple brin complémentaire de la première; et (b) à mettre en contact un échantillon (contenant la séquence de l'acide nucléique cible) avec la première et la deuxième sonde dans des conditions d'hybridation. Au moins une des sondes est normalement marquée par un marqueur détectable. L'amplification se termine par la fixation de la première sonde à la séquence cible par immobilisation de plusieurs marqueurs détectables par des fixations répétées et alternées de la deuxième sonde à la première, puis de la première à la deuxième, et ainsi de suite.
PCT/AU2008/001284 2007-08-30 2008-08-29 Méthode de détection d'un acide nucléique Ceased WO2009026651A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2007904702A AU2007904702A0 (en) 2007-08-30 A method of detecting a nucleic acid
AU2007904702 2007-08-30

Publications (1)

Publication Number Publication Date
WO2009026651A1 true WO2009026651A1 (fr) 2009-03-05

Family

ID=40386582

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2008/001284 Ceased WO2009026651A1 (fr) 2007-08-30 2008-08-29 Méthode de détection d'un acide nucléique

Country Status (1)

Country Link
WO (1) WO2009026651A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10557851B2 (en) 2012-03-27 2020-02-11 Ventana Medical Systems, Inc. Signaling conjugates and methods of use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046512A2 (fr) * 2001-11-23 2003-06-05 Royce Technologies Llc Procede permettant l'amplification de signaux de titrage biologique moleculaires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046512A2 (fr) * 2001-11-23 2003-06-05 Royce Technologies Llc Procede permettant l'amplification de signaux de titrage biologique moleculaires

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10557851B2 (en) 2012-03-27 2020-02-11 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
US11906523B2 (en) 2012-03-27 2024-02-20 Ventana Medical Systems, Inc. Signaling conjugates and methods of use

Similar Documents

Publication Publication Date Title
WO1999040224A1 (fr) Detection directe de l'arn au moyen d'une transcriptase inverse depourvue de fonction ribonuclease h
JPH05508074A (ja) インビトロにおける増幅を用いたポリヌクレオチド捕捉アッセイ
CN116829735A (zh) 检测靶核酸序列的方法
AU2015215750B2 (en) Nucleic acid detection or quantification method using mask oligonucleotide, and device for same
US8680257B2 (en) Kits for comparative transcript analysis and mutant sequence enrichment
WO2018003550A1 (fr) Ensemble d'amorces, trousse et procédé de détection d'au moins deux acides nucléiques cibles
WO2005019469A2 (fr) Detection et quantification d'arn
WO2006095550A1 (fr) Amorce de pcr, methode mettant en application cette amorce et produit amplifie par pcr, dispositif et complexe adn-proteine mettant en application ce produit amplifie par pcr
WO2009026651A1 (fr) Méthode de détection d'un acide nucléique
CN111549104A (zh) 一种circRNA驱动的基于长链DNA支架的DNA纳米带的制备方法及其肿瘤应用
US20020155456A1 (en) Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids
Cheng et al. Detection of multiple SNPs in numerous samples with polyacrylamide gel-based microarray
EP4590852A1 (fr) Procédé de détection d'une séquence d'acide nucléique cible
WO2006028162A1 (fr) Procédé de formation de polymère pour sonde de signal
EP2561098B1 (fr) Détection d'acides nucléiques à médiation par une enzyme captive
Wu et al. Detection DNA point mutation with rolling-circle amplification chip
US20100113298A1 (en) Detection of rna with micro-arrays
JP4560285B2 (ja) 核酸沈澱剤を利用した核酸増幅産物のハイブリダイゼーションアッセイ
WO2017006859A1 (fr) Procédé pour détecter un acide nucléique cible
US20030124604A1 (en) Method for amplification of molecular bio-assay signals
WO2022200213A1 (fr) Détection de l'activité de clivage et de ligature de topoisomérases d'adn
WO2025006625A1 (fr) Acides nucléiques imbriqués pour l'hybridation de cibles
WO2023170144A1 (fr) Procédé de détection d'une séquence d'acide nucléique cible
JP2023049233A (ja) 標的ｒｎａ検出方法及びプローブキット
CN117987389A (zh) 减少KASP基因分型非特异扩增的Taq DNA聚合酶及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08783029

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WPC Withdrawal of priority claims after completion of the technical preparations for international publication

Ref document number: 2007904702

Country of ref document: AU

Date of ref document: 20100222

Free format text: WITHDRAWN AFTER TECHNICAL PREPARATION FINISHED

122 Ep: pct application non-entry in european phase

Ref document number: 08783029

Country of ref document: EP

Kind code of ref document: A1