WO2009026651A1

WO2009026651A1 - A method of detecting a nucleic acid

Info

Publication number: WO2009026651A1
Application number: PCT/AU2008/001284
Authority: WO
Inventors: Aaron Ingham; Moira Menzies
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 2007-08-30
Filing date: 2008-08-29
Publication date: 2009-03-05
Anticipated expiration: 2010-02-28

Abstract

The present invention provides a method of amplifying a signal to detect a target nucleic acid comprising : (a) providing a first single stranded nucleic acid probe which possesses a first region complementary to part of the target, a third region substantially identical to the first region (in the same orientation) and (optionally) a second spacing region between the first and third, as well as a second single stranded nucleic acid probe complementary to the first, and (b) contacting a sample (with the target nucleic acid sequence therein) with the first and second probes under hybridisation conditions. Typically, at least one of the probes is labelled with a detectable label. Amplification is achieved by the first probe binding to the target sequence through immobilization of a plurality of detectable labels by repeated, alternated binding of the second probe to the first, and the first to the second and so on.

Description

A METHOD OF DETECTING A NUCLEIC ACID

Technical Field

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.

Background Art

The amplification of nucleic acids 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.

There are a variety of methods available to amplify specific nucleic acid molecules. One common reaction for amplifying a nucleic acid present at low concentrations is the polymerase chain reaction (PCR) which employs unique primers designed to amplify a specific DNA molecule. Briefly, PCR involves repeatedly (i) converting a double stranded target DNA to single stranded DNA by raising the temperature (ii) allowing the DNA-specific primers to anneal to the two complementary, disassociated templates and (iii) formation of DNA along the templates from the primers under the influence of a DNA polymerase.

Other amplification methods include; the ligase chain reaction (LCR) , nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR) , strand displacement amplification (SDA) , loop mediated isothermal amplification (LAMP) and rolling circle amplification (RCA) . Each of these methods leads to amplification of the target nucleic acid through repeated rounds of enzymatically catalysed nucleic acid synthesis. Despite the ability of these methods to amplify target nucleic acids to a similar magnitude, within the relatively short time frame of an hour, a number of shortcomings still exist. 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. When this procedure is used for the detection of nucleic acids 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. To overcome this, 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. In amplifying the target molecules by methods such as PCR, specificity issues arise from the amplified reaction products being dispersed in the reaction solution and not covalently attached to the target thereby resulting in a diffuse signal. The incorporation of more signal generating material is a simple procedure but still limited by the size of the probe and is often not a valid option as the most efficient labelling procedure has already been used. Methods designed for the amplification of the probe signal, once hybridised to the target, are based on either the biotin/streptavidin or tyramide systems. In the case of biotin/streptavidin a fourfold amplification is possible as every biotin molecule incorporated in the probe will subsequently bind 4 molecules of streptavidin. If the streptavidin molecule is conjugated with a signal generating molecule such as horseradish peroxidase (HRP) a 4 -fold increase results. In the tyramide system a hundreds fold amplification is possible. 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.

In WO2003/046512 there is described a non- enzymatic amplification (NEA) process which, as the name suggests, does not require any enzyme to amplify nucleic acid molecules, and can be used for signal amplification for in situ hybridisation.

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. Following hybridisation of the probe reference primer complex two amplification primers, designated amplifier I and amplifier II, are added to amplify the signal. With reference to Fig. 2 of WO 2003/046512 it can be seen that 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.

In operation the NEA method requires a first hybridisation step during which time the gene specific probe binds to the complementary target sequence, if present. However, 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.

It will be appreciated that 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. A further issue is that 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. Additionally, 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.In this process 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.

Accordingly there remains a need for a non- enzymatic method of detecting a nucleic acid which is effective, has a high amplification potential while not prone to the range of issues identified above including the production of false positives and/or incorrect imaging .

Summary of the Invention

According to a first aspect the present invention provides a method of amplifying a signal to detect a target nucleic acid, comprising the steps of:

(1) providing 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.

In an embodiment the first and the second single stranded probes are provided by denaturing a double stranded probe comprising the first and second strands.

In an embodiment the target nucleic acid is immobilised to a solid support.

In an embodiment the target nucleic acid hybridises with a capture oligonucleotide bound to the solid support.

In an embodiment the capture oligonucleotide is covalently bound to the solid support.

In an embodiment a plurality of target nucleic acids are immobilised such as by hybridisation to an array of capture oligonucleotides.

In an embodiment 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.

In an embodiment the target nucleic acid is localised within a biological sample. In an embodiment the biological sample is a tissue or cell preparation, for example a tissue section or fluid containing cells applicable to a slide.

In a further aspect of the present invention there is provided a method of in situ hybridisation imaging comprising the steps of:

(1) providing 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

(3) developing a signal from the detectable label to produce an image demonstrating the location of the target nucleic acid in the biological sample.

In an embodiment, 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.

It will be appreciated that 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. Thus 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.

Accordingly, in a further aspect the present invention provides a method of forming a nucleic acid complex, comprising the steps of:

(1) providing 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, and a second single stranded nucleic acid probe substantially complementary to the first single stranded probe;

(2) contacting the first and second probes and thereafter maintaining conditions appropriate for hybridisation; whereby the nucleic acid hybrid complex is formed through repeated, alternate binding of the second probe to the first probe and the first probe to the second probe. In an embodiment, 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.

In a further aspect, 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. In an embodiment a target nucleic acid is present, for example to locate or anchor the nucleic acid complex. In this embodiment the first region of the first single stranded nucleic acid probe is substantially complementary to sequence in a segment of said target nucleic acid.

In another aspect, 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.

In another aspect, 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.

In use, 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. Brief Description of the Drawings

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. In order to obtain a suitable signal from the conventional probe 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.

Detailed Description of the Invention

The invention relates to a method of amplifying a signal to detect hybridisation of a nucleic acid probe to a target nucleic acid. As used herein, "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) . In one embodiment, nucleic acid analogues are used which enhance hybridisation between complementary sequences. For example, 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). The methods described herein can be carried out with any of the 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. As used herein, a nucleic acid sequence is "substantially complementary" to another nucleic acid sequence if the two sequences are capable of hybridising. Typically, a nucleic acid sequence is substantially complementary to another nucleic acid sequence if greater than 85% of the sequence 1 "3

is capable of forming Watson-Crick base pairing with the other sequence, typically greater than 90% of the sequence, more typically greater than 95% of the sequence and even more typically 100% of the sequence. Sequences that are substantially complementary will hybridise under stringent conditions. Defining appropriate hybridization conditions is within the skill of the art. See eg. Sambrook et al . , DNA Cloning, vols. I, II and III. Nucleic Acid Hybridization. Typically, "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 5O⁰C, 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% Fiσoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 75OmM NaCl, 75mM sodium citrate at 42°C. An example of stringent conditions is hybridisation in 50% formamide, 4 X SSC (0.75M NaCl,

0.075M sodium citrate), 1 X Denhardt ' s solution, sonicated salmon sperm DNA (500μg/mL) , and 5% dextran sulfate at 42°C for 1 hour, and washes at 42°C in 1 X SSC and 52°C in 0.5 x SSC. As used herein "denaturation" and its equivalents refers to separating annealed nucleic acid molecules.

The third region of the probe contains sequence substantially identical to the first region and in the same orientation. Typically, 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. In an embodiment the first and third regions of the probe are identical in their sequence and orientation.

In an embodiment the 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. In one embodiment 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 .

In one embodiment, 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. Typically, 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 . In short, in this embodiment the probes are substantially complementary along their length and capable of hybridising throughout the length of the overhang.

In one aspect, the method comprises providing a first single stranded nucleic acid probe and a second single stranded nucleic acid probe. In one embodiment, the first and second single stranded nucleic acid probes are DNA strands. In another embodiment, the first and second single stranded nucleic acid probes are RNA strands. In another embodiment, the first single stranded nucleic probe is a DNA strand and the second single stranded nucleic acid probe is an RNA strand. In another embodiment, the first single stranded nucleic acid probe is an RNA strand and the second single stranded nucleic acid probe is a DNA strand.

In one embodiment, first and second single stranded nucleic acid probes are provided by denaturing a double stranded probe comprising the first and second strand. In another aspect, the method comprises providing a double stranded probe with a first strand and a second strand. In an embodiment the probe is a double stranded DNA probe. In another embodiment, the probe is a double stranded RNA probe. In another embodiment, 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.

Design of nucleic acids appropriate for use in the present invention is well within the capability of the person skilled in the art. Advantageously 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.

Amplification of Region 2

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.

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 5O⁰C 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. However, in this case 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. In addition, 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.

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.

Alternatively, 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. In one embodiment a probe may be labelled directly and used as a DNA probe. In another embodiment, 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. Cold Spring Harbor Laboratory Press, Plainview, N.Y.. 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. Ideally 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. For example 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. Likewise DIG may be detected through binding of a similar anti-DIG antibody-enzyme conjugate. In standard systems 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.

In preferred embodiments of the 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.

In an embodiment detecting a target nucleic acid involves visualising a nucleic acid which is known or expected to be present in the sample. In an embodiment the target nucleic acid is a mRNA and, in particular, a mRNA of relatively low abundance or one which is localised in a sample. In an embodiment the target nucleic acid is a DNA molecule.

It will be appreciated by persons skilled in the art that 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.

In an embodiment the method of the invention is used for in situ hybridisation. In this process 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.

In an embodiment the method of the invention is used for Southern, Northern or Dot blot hybridisation. In these processes 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.

In an embodiment 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. In particular, it is envisaged that 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.

In an embodiment the method is used for detecting polymorphisms or specific alleles.

It will be appreciated that 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. For example, in in situ hybridisation techniques it is desirable to visualise the nucleic acid in the location where it exists and, for example, photomicrographs such as in Figures 3 to 5 may be produced demonstrating the location of the target nucleic acid.

Equally, the target nucleic acid may be present in solution in a sample presented for analysis.

Accordingly, in an embodiment it is envisaged that the target nucleic acid may be immobilised to a solid support.

As used herein the term "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.

In an embodiment 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. In an embodiment the capture probe is chemically modified to allow covalent linkage to the solid support. Likewise, 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. Thus the linkage of the capture oligonucleotide takes place through reaction of the introduced group with a functional group on the solid support. For example, 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. The various chemistries which may be employed to covalently link an oligonucleotide to a surface are described, for example, in JK Veilleux and LW Duran, "Covalent Immobilisation of Biomolecules to Preactivated Surfaces", IVD Technology 2, No. 2 (1996:26-31), the contents of which are incorporated herein by reference. In an embodiment the surface of a solid support such as a polystyrene 96-well plate is coated with a layer of reactive N-oxysuccinimide esters which react with nucleophiles such as primary amines. The 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 .

Several mechanisms are available for adding a primary amine onto a nucleic acid. The most common method is to incorporate the amine onto either the 5¹ or 3¹ end of the molecule during synthesis. In an embodiment 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.

In an embodiment hapten linkage such as biotin- avidin or biotin-streptavidin systems may be employed. For example, 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.

In a further embodiment 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.

In an embodiment 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. Methods for preparing a microarray having a pre-selected array of immobilised oligonucleotides to capture target nucleic acid sequences from a sample are described, for example, in US Patent Publication 2008/0194413, the contents of which are incorporated herein by reference. A target nucleic acid may be the subject of a simple "on/off test" such as when immobilised to a dipstick. In this embodiment 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.

In an embodiment a nucleic acid lateral flow detector may be employed. In this embodiment 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.

In an embodiment there may be non-specific binding of target nucleic acids to a solid support. For example, 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 8O⁰C 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. For example 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.

The invention will now be described in detail by way of reference only to the following non-limiting examples .

Examples

Example 1 - Preparation of Probes for ovine MUC5, XDH, NOS2A, FGFlO and BMP4

The general approach for preparing probes for use in amplifying a signal to detect a target sequence is shown in Figure 1.

To prepare 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.

Table 1: Primer Pairs for generation of gene specific products

Gene Primer Primer Sequence Size of amplified fragment

MUC5 MUC5-F S'-GCTCCGCCTACGAGGACTTC-S' 97bp

MUC5-R 5'-GACCAGGCCGTCCAGCTT-S'

XDH XDH-F S'-AGCATCCCCACAGAGTTCAG-a' 197bp

XDH-R 5 ' -GGGCTATCTAGCCGGAAGAG-3 '

NOS2A NOS2A-F δ'-TCAGAGCCACGATCCTCTTT-S' 250bp

N0S2A-R 5 ' -CCGGAACTTGTTGGTGAGTT-3 '

FGFlO FGFlO-F S'-GATTGAGAACGGGAAGGTCA-S' 243bp

FGFlO-R δ'-CCATTGTGCTGCCAGTTAAA-S'

BMP4 BMP4 -F 5 ' -AGGGCATCGGTCTGGAGTAT-3 ' 235bp

BMP4 -R 5 ' -ATACGATGAAAGCCCTGCTC-3 ' These primers were used in a PCR consisting of 30 cycles of: 94⁰C for 30 seconds, 55⁰C for 30 seconds and 72⁰C for 30 seconds. Reactions were performed in 20 μl total volume and contained 10 ng of either ovine gut or ovine foetal skin cDNA, 0.9μM of specific primers and 1 Unit of Taq DNA polymerase in 1 x PCR buffer [45mM Tris.HCL (pH8.8); HmM (NH₄J₂SO₄; 4.SmM MgCl₂; 6.7mM 2- mercaptoethanol; 4.4μM 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.

To prepare region 1, a PCR was performed in a total of 50μl containing; 5ng of plasmid (containing gene specific region, generated above); 2.5 Units of Taq DNA polymerase; 1 x PCR buffer and 0.9μM 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 72⁰C for 30 seconds.

Table 2 : Primer pairs for generation of Region 1 Gene Primer Primer sequence Size of amplified fragment

MUC5 T7-MUC5 5' -TAATACGACTCACTATAGGG 135bp GCTCCGCCTACGAGGACTTC-3 '

Muc5-AR 5' -AGACAGGGGGCAGAGCGT GACCAGGCCGTCCAGCTT-3 '

XDH T7-XDH 5' -TAATACGACTCACTATAGGG 235bp AGCATCCCCACAGAGTTCAG-3 '

XDH-AR 5' -AGACAGGGGGCAGAGCGT GGGCTATCTAGCCGGAAGAG-3'

N0S2A T7-NOS2A 5' -TAATACGACTCACTATAGGG 288bp TCAGAGCCACGATCCTCTTT-3 '

NOS2A-AR 5' -AGACAGGGGGCAGAGCGT CCGGAACTTGTTGGTGAGTT-3 '

FGFlO T7 -FGFlO 5' -TAATACGACTCACTATAGGG 281bp GATTGAGAACGGGAAGGTC-3 '

FGFlO-AR 5' -AGACAGGGGGCAGAGCGT CCATTGTGCTGCCAGTTAAA-3 '

BMP4 T7-BMP4 5' -TAATACGACTCACTATAGGG 273bp AGGGCATCGGTCTGGAGTAT-3 '

BMP4-AR 5' -AGACAGGGGGCAGAGCGT ATACGATGAAAGCCCTGCTC-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 μl total volume and contained 200 ng of ovine genomic DNA, 0.9μM of specific primers and 1 Unit of Taq DNA polymerase in 1 x PCR buffer. PCR conditions involved 30 cycles of: 94⁰C 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 50μl of 1OmM Tris buffer. 0.5μl 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 50μl and incubated at 95°C for 5 minutes to denature the nucleic acid molecules. The solution was subsequently cooled to 5O⁰C for 30 seconds to allow annealing of region 1 to region 2 and incubated at 72⁰C for 15 minutes to extend the product, (see step 3 of Figure 1) . The gene specific T7 5'primer (eg T7-Muc5; see Table 2) and the 3' primer used to amplify region 2 (5'-CTGAATTCTGCAGGGAGAGG-S') were then added to the mix (0.9μM each) to amplify the product

(region 1 + region 2) . PCR conditions involved 30 cycles of: 94⁰C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds.

To prepare 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 50μl containing; 5ng of plasmid (containing gene specific region, generated above); 2.5 Units of Taq DNA polymerase; 1 x PCR buffer and 0.9μM of each gene specific primer pair listed in Table 2. PCR conditions were as follows, 30 cycles of: 94⁰C for 30 seconds, 55°C for 30 seconds and 72⁰C 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

MUC5 MUC5-SP6 δ'-ATTTAGGTGACACTATAGA 137bp

GACCAGGCCGTCCAGCTT-3 ' Muc5-B 5' -CCTCTCCCTGCAGAATTCAGC

GCTCCGCCTACGAGGACTTC-3 '

XDH XDH-SP6 5' -ATTTAGGTGACACTATAGA 237bp

GGGCTATCTAGCCGGAAGAG-3 ' XDH-B 5 ' -CCTCTCCCTGCAGAATTCAGC

AGCATCCCCACAGAGTTCAG-3 '

NOS2A NOS2A-SP6 δ'-ATTTAGGTGACACTATAGA 290bp

CCGGAACTTGTTGGTGAGTT-3 ' NOS2A-B S'-CCTCTCCCTGCAGAATTCAGC

TCAGAGCCACGATCCTCTTT-3 ' FGFlO FGFlO-SP6 δ'-ATTTAGGTGACACTATAGA 283bp

CCATTGTGCTGCCAGTTAAA -3' FGFlO-B 5'-CCTCTCCCTGCAGAATTCAGC GATTGAGAACGGGAAGGTCA -3' BMP4 BMP4-SP6 5' -ATTTAGGTGACACTATAGA 275bp

ATACGATGAAAGCCCTGCTC -3'

BMP4 -B 5 ' -CCTCTCCCTGCAGAATTCAGC

AGGGCATCGGTCTGGAGTAT -3 '

Amplification of Region 3 with the above primers resulted in the incorporation of a restriction site at the 5' end of the molecule which was the same as the restriction site at the 3' end of Region 2. Thus, to link Regions 1 and 2 to Region 3 , 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) .

Example 2 - In-Situ Hybridisation protocol using DIG labelled DNA probes

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.

Preparation of tissue slides

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 65⁰C for 15 minutes. The slides were subsequently rehydrated by washing the slide as follows:

Xylene 2 X 5 mxnutes

100% EtOH 2 X 5 minutes

90% EtOH 1 X 5 minutes

70% EtOH 1 X 5 minutes

H₂O 1 X 5 minutes

The washed slides were then treated with 500μl of 15μg/ml Proteinase K in Buffer I [10OmM Tris-HCl; 15OmM

NaCl; pH 7.5] for 10 minutes in humid conditions at 38⁰C.

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

0.4% cold formaldehyde for 5 minutes.

C. Hybridisation of tissue slides

Stabilised tissue slides were washed in 2 x SSC at room temperature for 5 minutes. Each slide was then pre-hybridised by incubated with 500μl hybridisation buffer [4xSSC; 50% formamide; Ix Denhardt's; 0.5mg/ml salmon sperm DNA; 5% Dextran sulfate] (minus probe) for 1 hour at 42⁰C] . During the prehybridisation step, 30ng of probe was denatured on a heat block at 95⁰C for 6 minutes, then place on ice for 1 minute. 500μl hybridisation buffer was also denatured on a heat block at 95⁰C for 6 minutes, then place on ice for 1 minute. The pre-hybridisation buffer was removed and 500μl of hybridisation buffer containing

30ng labelled DNA probe was added to the slide. The slide was incubated with the hybridisation mixture at 42⁰C in a humid chamber overnight .

Following hybridisation, the slides were washed in SSC at increasing stringency. Specifically, the slides were washed as follows:

2 x SSC for 15 minutes at room temperature 1 x SSC for 15 minutes at 42⁰C 0.5 x SSC for 15 minutes at 52⁰C

A. Developing the probe

To detect binding of the probe, 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 500μl blocking buffer II [0.5% blocking reagent (ROCHE) in buffer I] for 30 minutes at 38⁰C 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 500μl of anti-DIG antibody conjugate (1 in 2000) in blocking buffer II for 30 minutes at 38⁰C 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.

The slides were subsequently washed in buffer III [10OmM Tris-HCl; 10OmM NaCl; pH 9.5] at room temperature for 5 minutes. 500μl of colour development solution [0.5mg/ml NBT; 0.1875mg/ml BCIP; 5mM levamisole; 5OmM MgCl₂; 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; pHδ.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 H₂O, 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.

The effectiveness of a full length probe (comprising first second and third regions) was tested by comparing the results of in situ hybridisation using the full length probe to the results obtained using a conventional probe containing only a single region capable of hybridising to the target. Specifically, in situ hybridisation was carried out as described in Example 2, using the full length probe of Example 1 and a probe prepared in line with conventional protocols and corresponding to the first region only of the full length probe for the MUC5 gene. In addition, hybridisation was carried out using no probe or a probe corresponding to the second region only of the full length probe (the spacer region) as controls. The results of the hybridisation are shown in Figure 3.

As can be seen from Figure 3, hybridisation with the full length probe produced a signal that was significantly more intense than that of the conventional probe. Hybridisation with no probe, or a probe containing only the spacer region, gave only background staining. This result indicates that a probe in accordance with the invention is effective at producing an amplified signal in in situ hybridisation.

Example 4 - In situ hybridisation of ovine gut sections with XDH and NOS2A gene sequence.

To determine whether the use of a probe in accordance with the invention comprising a first, second and third region as described herein is effective for other genes, 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.

As can be seen from Figure 4, signal intensity from the full length probe was amplified significantly compared to the signal intensity from the conventional probe for both XDH and NOS2A.

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.

As can be seen from 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.

A 96-well plate with a polystyrene surface was primed with a capture oligonucleotide using the DNA-BIND^® (Corning Costar) system. DNA-BIND^® surface covalently binds to amine groups and will covalently immobilize aminated ssDNA by either the 5' or 3' end for hybridization.

Initially, lOOμl 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 4⁰C or for 1 hour at 37⁰C. 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 200μl of 3% BSA in Oligo Binding Buffer and the plate incubated for 30 minutes at 37⁰C.

A sample containing either target DNA or RNA, which is homologous (at the 3' region) to the capture oligonucleotide, can be hybridised to the DNA-BIND^® plate. For RNA transcripts, 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 95⁰C for 6 minutes then quickly transferring the tube to ice for 1 minute. For hybridisation, lOOμl/well of hybridisation solution containing the target nucleic acid was added and the plate was incubated for 60 minutes at a temperature that was 5⁰C (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 95⁰C for 6 minutes, then placed on ice for 1 minute. The probe was then added to the hybridisation solution and lOOμl added to each well.

Incubation for 60 minutes at the same temperature as the target nucleic acid hybridisation followed. After probe 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 .

For detection, lOOμl of blocking solution (3% BSA in maleate buffer) containing 1:2000 anti-DIG-AP antibody conjugate (ROCHE) was added followed by incubation for 30 minutes at 37⁰C. The wells were washed three times with maleate buffer. Finally, lOOμl of fresh substrate solution (p-nitrophenyl phosphate; SIGMA) was added to each well. The plate was then incubated at 37⁰C for 10, 20, 30, 40, 50 and 60 minutes and the OD (405nm) read. Signals should be above 0.6 while background should be well below 0.1.

Claims

1. A method of amplifying a signal to detect a target nucleic acid, comprising the steps of: (1) providing 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 probes 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.

2. The method of claim 1, wherein the first and the second single stranded nucleic acid probes are provided by denaturing a double stranded probe comprising the first and second strands.

3. A method as claimed in either one of claims 1 or 2 wherein the target nucleic acid is immobilised to a solid support .

4. A method as claimed in claim 3 wherein the target nucleic acid hybridises with a capture oligonucleotide bound to the solid support.

5. A method as claimed in claim 4 wherein the capture oligonucleotide is covalently bound to the solid support.

6. A method as claimed in any one of claims 1 to 5 wherein a plurality of target nucleic acids are immobilised.

7. A method as claimed in any one of claims 1 to 5 wherein 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.

8. A method as claimed in either one of claims 1 or 2 wherein the target nucleic acid is localised within a biological sample.

9. A method as claimed in claim 8 wherein the biological sample is a tissue sample.

10. A method of in situ hybridisation imaging comprising the steps of:

(1) providing 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 comprising regions substantially complementary to the first single stranded probe, the first probe and/or the second probe being labelled with a detectable label;

(3) contacting a sample in which the target nucleic acid is localised with the first and second probe and thereafter maintaining conditions appropriate for hybridisation; and (4) developing a signal from the detectable label to produce an image demonstrating the location of the target nucleic acid in the tissue or cell preparation.

11. The method of claim 10, wherein 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.

12. A method of forming a nucleic acid complex, comprising the steps of:

(2) contacting the first and second probes and thereafter maintaining conditions appropriate for hybridisation; whereby the nucleic acid hybrid complex is formed through repeated, alternate binding of the second probe to the first probe and the first probe to the second probe.

13. The method of claim 12, wherein 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.

14. 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 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.

15. 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.

16. 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.