NO844849L - HYBRIDIZATION ANALYSIS USING LABELED COUPLES OF HYBRID BINDING REAGENTS - Google Patents

Description

The present invention relates to nucleic acid hybridization analysis methods and reagent systems for detecting specific polynucleotide sequences. The principle of nucleic acid hybridization assays was developed by work in the field of recombinant DNA research to determine and isolate particular polynucleotide base sequences of interest. It was found that single-stranded nucleic acids, e.g. DNA and RNA, as obtained by denaturing the double-stranded forms, hybridize or recombine under suitable conditions with complementary single-stranded nucleic acids.

By labeling such complementary probe nucleic acids with a

easily detectable group, one can detect the presence of any polynucleotide sequence of interest in an experimental medium containing sample nucleic acids in single-stranded form.

In addition to the field of recombinant DNA, the analytical hybridization technique can be used for the detection of important polynucleotides, among other things, in the fields of human and veterinary medicine, agriculture and food science. In particular, the technique can be used for the detection and identification of etiological agents such as e.g. bacteria and viruses, to examine bacteria with regard to antibiotic resistance, to facilitate the diagnosis of genetic disorders such as sickle cell anemia and thalassemia, and for the detection of cancer cells. A general overview of the technique and its present and future importance, found in Biotechnology (August 1983), pages 471-478.

The following information is provided to make known information believed to be relevant to the present invention. The following information does not constitute prior art to be assessed against the present invention.

The state of the art in nucleic acid hybridization analysis techniques generally includes a separation of hybridized and unhybridized labeled probe. This necessary separation step is facilitated by either the sample nucleic acids or the nucleic acid probe being immobilized on a solid support. Typically, the hybridization between particular base sequences or genes of interest in sample nucleic acids and a labeled form of the probe nucleic acid is detected by separation of the solid support from the remaining reaction mixture containing unhybridized probe, followed by detection of the label on the solid support.

Attempts are constantly being made to simplify the analytical procedure for performing nucleic acid hybridization analyses. The primary purpose of these efforts is to reduce the complexity of the procedure to such an extent that it can be easily and routinely performed in clinical laboratories. The necessity of a separation step is largely an obstacle to these endeavours. The separation step requires considerable expertise if it is to be performed in an analytically reproducible manner, and it is a process that cannot be easily automated or applied to large trial volumes.

Furthermore, two significant difficulties are encountered with conventional methods that include immobilization of sample nucleic acids. First, the procedures required to achieve immobilization are generally time-consuming and constitute an additional step which is undesirable for routine use of the technique in clinical laboratories. Second, proteins or other materials in the heterogeneous sample, especially in the case of clinical samples, can affect the immobilization process.

As alternatives to the immobilization of sample nucleic acids

and addition of the labeled probe, one can immobilize a probe and label the nucleic acids in situ, or one can use a dual hybridization technique that requires two probes, one of which is immobilized and the other labeled. However, the first option is even less desirable because in

in situ labeling of sample nucleic acids requires greater technical skills than can be expected from clinical laboratory personnel, and there are no simple, reliable methods to control the labeling yield, this can be a significant problem if the labeling medium contains variable amounts of inhibitors for the labeling reaction. The dual hybridization technique has the disadvantage that it requires an additional reagent and an incubation step, and the kinetics of the hybridization reaction can be slow and inefficient. The accuracy of the analysis can also be variable if the complementarity of the two probes with the sample sequence is variable.

Some of the problems discussed above are solved by

using an immobilized RNA probe and detecting the resulting immobilized DNA'RNA or RNA'RNA hybrids with a labeled specific anti-hybrid antibody (see US Patent Application No. 616,132, filed June 1, 1984). This technique still requires a separation step and therefore has the disadvantages discussed above, and is common to all hybridization techniques that require a separation step.

US-PS 3,996,345; 4,233,402; and 4,275,149 describe assays for the detection of antigens by immunoassay techniques involving enzyme pairs and energy transfer interactions.

European Patent Application No. 70,685 proposes a hybridization assay technique that eliminates the need to physically separate hybridized from unhybridized probe. It is proposed to use a pair of probes that hybridize to adjacent regions of a polynucleotide sequence of interest, and to label a probe with a chemiluminescent catalyst such as e.g. the enzyme peroxidase and the other with an absorbing molecule for the chemiluminescent emission. The catalyst and absorbent labels must be placed close to the adjacent terminal ends of the respective probes in such a way that, upon hybridization, quenching of the chemiluminescent emission by energy transfer to the absorbing molecule is observed.

To perform such an analysis, one must be able to controllably synthesize two critical probe reagents so that the respective labels are brought into a quenching orientation with respect to the probe nucleic acid without affecting the tendency of the respective labeled probe segments to actually undergo hybridization.

One has now arrived at a nucleic acid hybridization assay which is based on the use of neighboring pairs of interacting labels which give a detectable signal that is measurably different for the hybridized probe compared to the unhybridized probe. In this way, there is no need to separate hybridized and unhybridized probe, this greatly facilitates the execution and automation of the analysis. In addition, the analysis signal is non-radioisotope in nature and thus fulfills another criterion for an appropriate analysis, the use of detection systems that do not include radioactivity.

According to the present invention, specific polynucleotide sequences are detected in a sample by forming a hybrid between the sequence to be detected and a nucleic acid probe that has a complementary sequence in such a way that the resulting hybrid has binding positions for two different specific binding reagents. One of these binding reagents includes a first label and the other a second label, where the interaction between the two labels provides a detectable response that is measurably different, either in a positive or negative sense, when the two labeled reagents bind to the same hybrid compared provided that they are not bound in this way.

When they tie the same hybrid, the two marks are brought

in neighboring interaction distance to each other, thereby increasing the interactions between the two labels that give a signal to a large extent compared to the relatively less frequent interactions that take place in the bulk solution between free diffusible, labeled reagents.

A preferred interaction between the two labels is a stepwise interaction where the first label participates in a first chemical reaction, so that a diffusible product is formed which is a participant in a second chemical reaction with the other label, so that a detectable product is formed . It is particularly preferred that the first and second brands are catalysts, e.g. enzymes, for the first and the second chemical reaction respectively. If e.g. the first brand is glucose oxidase and

the other being peroxidase, the hydrogen peroxide formed by the action of glucose oxidase on glucose will diffuse to the peroxidase label and be detected as an optical signal in the presence of a suitable indicator dye composition. Another preferred label pair is a pair involving energy transfer interaction such as between a fluorescent or luminescent substance, and a quencher of the photoemission from the first mark.

With regard to the nature of the specific binding agents

which is used, in principle at least one of the two must be bound to a position that is unique for hybrids formed in the analysis compared to unhybridized nucleic acids because the signal generation must be dependent on the formation of such a hybrid. Therefore, the specific binding reagent will preferably be an antibody that is selective for the hybrid compared to single-stranded nucleic acids present in the assay mixture. A wide variety of other binding reagents can be used as described below.

The present invention is characterized by a number of significant advantages. In addition to the main advantage which is the elimination of the separation step, no immobilization of either sample or probe nucleic acids is required, which gives rise to non-specific binding and reproducibility problems with commonly used hybridization techniques. Another advantage is that the analysis can be carried out without a washing step. The analysis reagents can be added step by step to the hybridization medium, without the need to wash insoluble carrier materials.

Another essential feature of the present invention is

that the detection system used is particularly effective because double-stranded duplexes can contain many binding positions for the first and the second labeled reagent. This results in large amounts of the first and second label being attached in an interacting configuration per unit hybridized probe. When considering antibodies to RNA"-DNA or RNA'RNA hybrids, a labeled antibody can bind to approximately every 10 base pairs of the hybrid. If the probe is, for example, 500 bases long, 40-50 antibodies can bind. Suitable ligands to labeled binding proteins, can be introduced into probes in amounts of 1 ligand per 5-20 bases.The presence of multiple binding positions on hybrids can

advantageously used when small amounts of hybrid must be detected.

Fig. 1-3 are schematic illustrations of preferred methods for carrying out the present invention.

These procedures are described in detail below.

The application of nucleic acid hybridization as an analytical tool is fundamentally situated on the double-stranded, duplex structure of DNA. The hydrogen bonds between the purine and pyimidine bases in the respective chains in double-stranded DNA can be reversibly broken. The two complementary single chains of DNA resulting from this "melting" or "denaturation" of DNA will associate (often called recombination or hybridization) so that the duplex structure is again formed. As is known, contact of a first single-stranded nucleic acid, either DNA or RNA, which includes a base sequence sufficiently complementary to (ie "homologous to") a second single-stranded nucleic acid under appropriate conditions will result in the formation of DNA'DNA-, RNA "DNA or RNA'RNA hybrids, depending on the starting materials.

The probe will include at least one single-stranded base sequence

which is essentially complementary to, or homologous to

with the sequence to be detected. However, such a base sequence need not be a single continuous polynucleotide segment, but may consist of two or more individual segments which are interrupted by non-homologous sequences. These non-homologous sequences can be linear, or

they can be self-complementary and form hairpin loops.

In addition, the homologous region of the probe may be flanked at the 3'- and 5<1->termini by non-homologous sequences, such as e.g. those which include the DNA or RNA of a vector into which the homologous sequence has been cut for propagation. In any case, the probe, when present as an analytical reagent, will show detectable hybridization at one or more points with the sample nucleic acids of interest. Linear or circular single-stranded polynucleotides can be used as a probe element, where larger or smaller parts are duplexed with a complementary polynucleotide chain or chains, provided that the critical homologous segment or segments are in single-chain form and available for hybridization with sample DNA or RNA. It is generally preferred to use probes that are essentially in single chain form. Preparation of a suitable probe for a particular assay is a matter of routine skill.

According to the principle of the present invention, the hybridization, which is dependent on the presence of the polynucleotide sequence of interest, and binding of the labeled reagents to the hybrid, results in the two labels being brought sufficiently close to each other so that the signal generated is interaction between them, measurably different from the signal formed by encounters during simple diffusion in the bulk analysis medium. Various interaction phenomena can be used in the present invention. The interaction can be chemical, physical, or electrical, or combinations of these forces. The environment for the label formed by hybridization and binding of the labeled reagents to the hybrids formed, hereinafter referred to as environment associated hybrid, must in at least one critical respect be distinctly different from the bulk medium. Since the hybridization determines the proportion of labels formed in localized hybrid environments compared to the bulk phase, the resulting signal response will depend on the presence of the sequence to be determined in the assay medium.

A preferred interaction between the two labels involves two chemical reactions where one label participates in the first reaction and forms a diffusible intermediate that participates in the second reaction with the second label,

and gives a detectable product. The microenvironment for the bound hybrid will consequently contain a higher localized concentration of the intermediate, so that the rate of the signal-producing second label reaction is increased, than the bulk solution. The two labels can respectively participate in the reaction as reactants or, particularly preferred,

as catalysts. Any catalysis can be either enzymatic or non-enzymatic.

A large number of different enzymes and catalysts can be used in the present invention. Useful enzymes for the first reaction include oxidoreductases, especially those containing nucleotides such as nicotinamide a de ni'n dinucleotide (NAD) or its reduced form (NADH), or adenosine triphosphate (ATP) as cofactors or those that produce hydrogen peroxide or other small diffusible products. Some examples are alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase and uricase. Other classes of enzymes such as hydrolase, transferase, lyases, and isomerases can also be used.

It is particularly preferred that the other brand is also

an enzyme where the product of the first enzyme reaction is a substrate or a cofactor for the second enzyme, so that rapid enzyme reaction is achieved in the environment of the bound hybrid. If e.g. the first enzyme is an oxidoreductase, which forms hydrogen peroxide as a product,

the second enzyme may be peroxidase. A detailed listing and description of useful enzymes can be found in US-PS 4,233,02 and 4,275,149. Other useful systems include an enzyme as the first label that catalyzes a reaction that forms a prosthetic group for an apoenzyme or proenzyme. Non-enzymatic catalysts can also serve as labels as mentioned earlier. As an example, reference can be made to US-PS 4,160,645.

Another preferred interaction between the tags is energy transfer interaction. The first mark is a photo-emitting substance such as a fluorescent or luminescent substance, the former will emit upon irradiation and the latter will emit upon chemical reaction. The photoemission can be absorbed by the other label, so that the emission is either clustered or provides another emission such as e.g. if the absorbent label itself is fluorescent. Pairs of compounds useful for this purpose are described in detail in US-PS 4,275,149 and 4,318,981. Some preferred fluorescent/quenching pairs are naphthalene/anthracene, α-naphthylamine/dansy1, tryptophan/dansyl, dansyl/fluorescein, ffluorescein/rhodamine, tryptophan/fluorescein, N-/p-(2-benzoxazolyl)phenyl/maleimide (BPM)/ thiochrome, MPB/8-anilino-1-naphthalenesulfonate (ANS), thiochrome/N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM), and ANS/DDPM. Some preferred luminescent/quenching pairs are luminol with fluorescein, eosin or rhodamine S.

If desired, various modifications of the labeled reagents can be used within the scope of the present invention. In a variation, one of the labeled reagents may include a solid phase to which the binding substance is bound and separately also labeled. The solid phase can be the walls of the reaction container or a dispersed solid such as e.g. polyacrylamide or agarose beads. One can also modify the binding substance with a bindable ligand, such as a hapten or biotin, and introduce label by adding a labeled anti-hapten antibody or labeled avidin before or after the hybridization reaction. It therefore appears that the mark can be connected to the binding substance directly or indirectly by means of intermediate components.

A critical feature of the present invention is the formation of a hybrid between the probe and the polynucleotide sequence of interest that includes binding sites for the two specific binding reagents. A principle for the analysis is 2.t the hybridization of the probe with the desired sequence means that the respective binding positions for the two binding reagents are brought to such a distance from each other that interaction between the labels can take place. Any design of the system that provides a binding position for at least one of the two labeled reagents that is unique to the hybrid can be used. This ensures that the desired localization of the tag pair will only take place during the formation of the hybrid.

The binding of labeling reagent to the hybrid will normally include a very specific non-covalent bond, which is characteristic of a number of biologically derived substances, especially binding proteins such as e.g. immunoglobulins. A variety of binding agents can be used to provide at least one binding reagent that has unique binding affinity for the hybrid and negligible binding affinity for single-stranded nucleic acids such as e.g. unhybridized probe and unhybridized sample- . nucleic acids.

Particularly preferred binding agents are antibody reagents

which have anti-hybrid binding activity and may be whole antibodies or fragments thereof, or aggregates or conjugates thereof, of conventional polyclonal or monoclonal type. Preferred antibody reagents will be those which are selective for binding to (i) DNA'RNA or RNA'RNA hybrids, or (ii) intercalation complexes. It is now known that antibodies can be stimulated so that they are selective

for DNA"RNA or RNA'RNA hybrids rather than single-stranded nucleic acids, at the present time, however, it is considered impossible to generate such selectivity in the case of DNA * DNA hybrids. To the extent that selective DNA * DNA antibodies are developed in the future, they will clearly be applicable of the present invention. Antibodies to DNA"RNA or RNA'RNA hybrids can be used where the probe is RNA and the test nucleic acids are DNA or RNA, or where

where the probes are DNA and the sample RNA.

It should also be noted that when referring to an RNA probe, used with an anti-DNA<*>RNA or anti-RNA<*>RNA reagent, it is meant here that not all the nucleotides included in the probe must be ribonucleotides, i.e. bear a 2'-hydroxyl group. The fundamental feature of an RNA probe used here is that it is sufficiently non-DNA

in nature to enable the stimulation of antibodies to DNA * RNA or RNA"RNA hybrids that include an RNA probe that does not cross-react to an analytically significant degree with the individual single chains that make up such hybrids. Therefore, one or more of the 2<1->positions may on the nucleotides included in the probe, be in the deoxy form, provided that the antibody-binding properties necessary for the present assay to be carried out are maintained to a significant extent. Similarly, an RNA probe may, in addition to, or alternatively to, such limited 2 '-deoxy modification, including nucleotides having other 2<1> modifications, or generally any modification along the ribose phosphate backbone, provided that it does not significantly affect the specificity of the antibody to the double-stranded hybridization product over its individual single chains.

Where such modifications are present in an RNA probe, the immunogen used to create the antibody reagent will preferably include one chain that has essentially corresponding modifications and the other chain will consist mainly of unmodified RNA or DNA, depending on whether the sample RNA or -DNA is to be detected. Preferably, the modified chain in the immunogen should bear identical to the modified chain in the RNA probe. An example of an immunogen is the hybrid poly(2'-0-methyladenylic acid)"poly (2<1->deoxythymidylic acid). Another example is poly(2'-0-ethylinosinic acid)"poly(ribocytidylic acid). The following compounds are further examples of modified polynucleotides that can be included in an RNA probe: 2'-O-methylribonucleotide, 2'-O-ethylribonucleotide, 2<1->acidodeoxy ribonucleotide, 2'-chlorodeoxyribonucleotide, 2'-0 -acetyl ribonucleotide, and the methylphosphonates or phosphorothiolates of ribonucleotides or deoxy ribonucleotides. Modified nucleotides may be present in RNA probes as a result of introduction during enzymatic synthesis of the probe from a template. E.g. are adenosine 5'-0-(1-thiotriphosphate) (ATpaS) and dATPaS substrates for DNA-dependent RNA polymerases and DNA polymerases, respectively. Alternatively, the chemical modification can be introduced after the probe has been produced. E.g. an RNA probe can be 2<1->0-acetylated with acetic anhydride under mild conditions in an aqueous solvent.

Immunogens for stimulation of antibodies specific for RNA-DNA hybrids may include homopolymeric or heteropolymeric polynucleotide duplexes. Among the possible homopolymeric duplexes, poly(rA)'poly(dT) is particularly preferred. /Kitagawa and Stollar (1982) Mol. Immunol. 19:413/. In general, however, heteropolymeric duplexes are preferred and these can be prepared in a number of different ways, including transcription of <t>X174 virion DNA with RNA polymerase /Nakasato (1980) Biochem 19:2835/. The selected RNA' DNA duplexes are adsorbed on a methylated protein, or otherwise bound to a conventional immunogenic carrier material, such as e.g. bovine serum albumin, and injected into the desired host animal /see also Stollar (1980) Meth.Enzymol. 70:70/. Antibodies to RNA'RNA duplexes can be created against double-stranded RNA from viruses such as e.g. reovirus or Fiji disease virus which infects e.g. sugar cane. Also homopolymeric duplexes such as e.g. blue. poly(ri)"poly (rC) or poly(rA)"poly(rU), can be used for immunization as above. Additional information regarding antibodies to RNA"DNA or RNA"RNA hybrids is provided in US Patent Application No. 616,132, filed June 1, 1984.

Antibodies to intercalating complexes can be raised against an immunogen which will usually comprise an ionic complex between a cationic protein or protein derivative (eg methylated bovine albumin) and the anionic intercalating nucleic acid complex. Ideally, the intercalator should be covalently linked to the double-stranded nucleic acid. Alternatively, the intercalator nucleic acid conjugate may be covalently linked to a carrier protein. The nucleic acid portion of the immunogen may include the specific paired sequences found in assay hybrids or may include any other desired sequence since the specificity of antibodies will generally not depend on the particular base sequence included. Additional information regarding antibodies to intercalation complexes is provided in US Patent Application No. 560,429, filed December 12, 1983.

As indicated above, the antibody reagent may consist of whole antibodies, fragments of antibodies, polyfunctional antibody aggregates, or generally any substance that includes one or more specific binding positions from an antibody. When in whole antibody form, they may belong to any of the classes or subclasses of known immunoglobulins, e.g. IgG, IgM etc. Any fragment of any such antibody that retains the specific binding affinity for the hybridized probe may also be used, e.g. the fragments of IgG conventionally known as Fab, F(ab') and F(ab 1)2• 1 in addition, aggregates, polymers, derivatives and conjugates of immunoglobulins or fragments thereof, can be used where appropriate

appropriately.

The immunoglobulin source for the antibody reagent can be obtained

by any means available such as by conventional antiserum and monoclonal techniques. Antiserum can be obtained by well known techniques which include immunization of an animal, such as a mouse, rabbit, guinea pig or goat, with a suitable immunogen. The immunoglobulins can also be obtained by somatic cell hybridization techniques, these result in what are usually referred to as monoclonal antibodies, this also involves the use of a suitable immunogen.

In those cases where an antibody reagent selective for intercalation complexes is used as one of the binding reagents, a number of intercalator compounds can be used. In general, it can be said that the intercalator compound is preferably a molecule of low molecular weight, which is planar, usually aromatic but sometimes polycyclic, and capable of forming bonds with double-stranded nucleic acids, e.g. DNA"DNA, DNA"RNA, or RNA"RNA duplexes, usually by insertion between base pairs. The primary binding mechanism is usually non-covalent, where covalent binding may occur as a second step where the intercalator has reactive or activatable chemical groups that will form covalent bonds with neighboring chemical groups on one or both of the intercalated duplex chains. The result of the intercalation is that neighboring base pairs are spread to a distance that is approximately twice the normal separation distance, this appears to be an increase in the molecular length for the duplex. Furthermore, a winding of the double helix of about 12-36° must take place for the intercalator to find its place. General overviews and further information can be found in Lerman, J. Mol. Biol. 3:18 (1961); Bloomfield et al ., "PhysicalChemistry of Necleic Acids", ch. 7, pp. 429-476;Harper

and Rowe, NY (1974); Waring, Nature 219:1320 (1968); Hartmann et al., Angew. Chem., Engl. Oath. 7:693 (1968); Lippard, Accts. Chem. Res. 11:211(1978); Wilson, Intercalaltion

Chemistry (1982), 455; and Berman et al., Ann. Fox. Biophys. Bio meadow. 10:87 (1981); as well as the previously mentioned US patent application no. 560,429. Examples of intercalators are acridine dyes, e.g. acridine orange, phenanthridines, e.g. ethidium, phenazines, furocoumarins, phenothiazines, and kino-lines.

The intercalation complexes are formed in the assay medium during the hybridization using a probe that is modified in its complementary, single-stranded region so that it has the intercalator chemically bound to it in such a way that the intercalation complexes are formed during the hybridization.

Any suitable method may be used

to achieve such binding. A particularly useful method involves the acid intercalator. When exposed to high-wavelength ultra-violet light or visible light, the reactive nitrenes are readily generated. The nitrenes of aryl azides prefer insertion reactions to conversion products / see White et al., Methods in Enzymol. 46: 644 (1977]_/. Examples of azido intercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazides, ethidium diazod, ethidium dimer-azide /Mitchell et al., JACS 104:4265 (19 8-2 )_/, 4 -azido-7-chloroquinoline, and 2-azidofluorene. Other useful photoreactive intercalators are the furocoumarins which form /2+2/ cycloaddition products with pyrimidine residues. Alkylating agents can also be used, such as bis-chloroethylamines and epoxides or aziridines , e.g., aflatoxins, polycyclic hydrocarbon epoxides, mitomycin, and norphillin A. The intercalator-modified duplex is denatured

so that it gives the modified single-stranded probe.

As mentioned, at least one of the bindable reagents must be off

the type discussed above, however, there is greater choice for the second binding reagent since it does not need to discriminate between the hybrid and other nucleic acids, including the probe and sample materials, to achieve the desired effect. Consequently, the other can be binding

the reagent be the same as or a different binding material than that used in said reagent, except that when it is the same, the final difference consists in the assay reagent being labeled with the other label. When using an antibody reagent that is selective binding of intercalation complexes as both binding reagents, one normally wants to use a probe that has been modified so that it has the intercalator chemically bound to the probe,

as opposed to additive as a soluble compound.

Alternatively, the second binding reagent can be any material that binds to the probe. That is that it can be an antibody reagent that binds non-specifically to nucleic acids such as e.g. anti-DNA"DNA. In another embodiment, a probe is used which includes a specifically binding ligand part where the non-specific binding reagent is a binding partner for such a ligand part. In such a case, the ligand part/binding partner can -the pair is selected from known materials such as haptens/antibodies, antigens/antibodies, lecithins/polysaccharides, hormones/receptor proteins, etc. In this connection, reference can be made to British patent 2,019,408, European patent application 63,879 and 97,373 and PCT published application 83-1459 and 83-2286.

Although it is generally preferred that the critical microenvironments for the bound hybrids are created in part or all of the hybridized complementary region of the probe/probe duplex, in principle such microenvironments can also be found at the boundary between such a complementary region and the flanking region. This can be done by using binding reagents as described above. In addition, one can use a binding substance for a binding position on double chain flanking regions of the probe as the non-specific binding reagent. Examples of such binding site/binding partner pairs are promoter sequences (eg, lac promoter, trp promoter, and promoters associated with bacteriophages)/polymerase; lac operator/lac repressor protein; and antigenic nucleotides and sequences (eg, Z-

DNA and rare nucleotides such as 5-halodeoxyuridines)/ antibodies thereto.

With reference to the drawings and the examples that follow, a couple of specific embodiments of the present analysis method can be described. In the method shown in fig. 1, an unmodified polynucleotide probe is used which is either RNA or DNA when the sample sequence of interest is RNA, when the sample sequence is DNA the probe is RNA. The binding reagents are the same or different antibody reagents (Ab) that are selective for RNA'DNA or RNA'RNA hybrids depending on which are present. One antibody reagent is labeled with glucose oxidase (GOD) and the other more peroxidase (POD). In the presence of glucose and a hydrogen peroxide color indicator, the rate of color development will depend on the extent of hybridization that takes place. In the bound hybrid, glucose oxidase and peroxidase are brought to a distance a from each other, this results in an increased rate of color development due to rapid diffusion of hydrogen peroxide to peroxidase . In the bulk medium, hydrogen peroxide must diffuse the average distance b to cause a color development that gives rise to a significantly altered rate. Optionally, catalase may be included in the bulk solution in concentrations sufficient to effectively destroy any hydrogen peroxide that diffuses away from the microenvironment for the bound the hybrid, so that the background color e formation is reduced.

The use of a ligand-modified probe is shown in fig.

2, where biotin (Bio) is the selected ligand. In this case, the GOD-labeled binding reagent is again an antibody to RNA'DNA or RNA'RNA, whichever is present, and the other binding reagent is POD-labeled avidin (Av). The registration of the color development will take place as described in procedure A.

In the method shown in fig. 3, the probe is doubly modified, with both biotin as in method B and a chemically bound intercalator (I). An antibody to intercalation complexes involving I is labeled with a fluorescent substance

(F) and avidin is labeled with a quencher (Q). Fluorescence

and the extinguisher marks are brought into energy transfer distance a

in the bound hybrid, so that upon irradiation with light of a first wavelength (hv^), the emitted energy (h^) will be absorbed by the quencher and not detected. On the other hand, fluorescently labeled antibody in the bulk solution will be at an average distance b from the quencher, which is not close enough for efficient energy transfer,

and the fluorescence emission is observed. The amount of light detected is inversely proportional to the extent of hybridization that takes place.

The sample to be analyzed can be any medium of interest, and will usually be a liquid sample of medical, veterinary, environmental, nutritional, or industrial importance. Human and animal samples and body fluids can in particular be analyzed by the present method, including urine blood (serum or plasma), amniotic fluid, milk, cerebrospinal fluid, saliva, feces, lung aspirates, throat swabs, genital swabs and exudates, rectal swabs, and nasopharyngeal aspirates. In cases where the sample taken from the patient or other sources for examination mainly contains double-stranded nucleic acids, as they e.g. contained in cells, the sample is processed to denature the nucleic acids,

and, if necessary, first to release nucleic acids from cells. Denaturation of nucleic acids is preferably carried out by heating in boiling water, or by alkali treatment (e.g.

0.1 N sodium hydroxide) which, if desired, can also be used to light the cells. Release of nucleic acids can also be achieved by e.g. mechanical degradation (freezing/thawing, abrasion, with the help of sound waves), physical/chemical degradation (cleaning agents such as "Triton", "Tween", sodium dode-

cyl sulfate, alkali treatment, osmotic shock, or heat), or enzymatic lysis (lysozyme, proteinase K, pepsin).

The resulting test medium will contain nucleic acids in single-chain form, which can then be analyzed according to the present hybridization method. In cases where RNA'DNA is to be detected with labeled antibody reagents, m RNA can

and rRNA in the sample is prevented from participating in the binding reaction by conventional methods such as e.g. treatment under alkaline conditions, e.g. the same conditions used to denature the nucleic acids in the sample.

As is known, different hydridation conditions can be used in the analysis. Typically, the hybridization will take place at somewhat elevated temperatures, e.g. between 35 and 75°C, usually around 65°C, in a solution including buffer at pH between approx. 6 and 8, and with suitable ionic strength (e.g. 5XSSX where 1XSSC = 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0). In cases where it is desirable to use lower hybridization temperatures, hydrogen-binding reagents such as e.g. dimethylsulfoxide and formamide are included. The degree of complementarity between the sample and probe strands required for hybridization to occur depends on the stringency of the conditions. Factors that determine stringency are well known.

Normally, the temperature conditions chosen for hybridization will be incompatible with the binding of the anti-hybrid reagent to the hybrids formed and detection of the label response. Accordingly, the anti-hybrid binding step and the label detection step will take place after the hybridization step is completed. The reaction mixture is normally brought to a temperature in the range from approx. 3°C to approx. 40°C, and the binding and detection steps are then performed. Dilution of the hybridization mixture before adding the antibody reagent is desirable when the salt and/or formamide concentrations are high enough to significantly affect the antibody binding reaction.

The present invention additionally provides a reagent system, i.e. a reagent combination or devices,

which includes all the essential elements required to carry out a desired analysis procedure. The reagent system is in commercially packaged form, as a composition or additive where the compatibility of the reagents permits, in an experimental configuration,

or usually as a trial package, i.e. a packaged combination of one or more containers, devices etc. containing the necessary reagents, and usually including written instructions regarding the performance of the analyses. The reagent systems according to the present invention include all configurations and compositions for carrying out the various hybridization methods described here.

In all cases, the reagent system will include (1) a nucleic acid probe as described, and (2) first and second labeled binding reagents. A test package form of the system can also include auxiliary chemicals such as e.g. the components of the hybridization solution and denaturants capable of converting double-stranded nucleic acids in a sample to single-stranded form. Preferably, a chemical lightening and denaturing agent is included, e.g. alkali, for treating samples so that single-stranded nucleic acids are released.

The present invention shall now be illustrated, but not limited, by the following examples.

Example 1.

Hybridization assay for bacterial RNA controlled with labeled antibodies to RNA"DNA hybrid.

A. Monoclonal antibody to RNA'DNA hybrid is created

following the procedure described by Stewart et al., (1981)

PNAS 78, 3751. The antibody is isolated from peritoneal fluid

by affinity chromatography on protein A "Sepharose".

About. 1 ml peritoneal fluid per ml protein A "Sepharose" is added to the column and then the column is washed with 50 millimolar (mM) bicine buffer, pH 8.0, 0.15 molar (M) NaCl until the absorbance of the effluent at 280 nanometers (mn) is less than 0.05 . The bound antibody is eluted with 0.1 M glycine buffer, pH 3.0, and antibody is detected by measuring the absorbance at 280 nm. The collection of antibody is set to

pH 5.0 or higher with the addition of IM bicine buffer, pH

8.5. The collection is dialysed in 0.1M borate buffer, pH 9.0.

The antibody is readily labeled with 5-(4,6-dichlorotriazin-2-yl)-aminofluorescein (DTAF) (Molecular Probes, Inc., Junction City, Oregon) by the method of Blakeslee and Baines

(1976) J. Immunol. Meth. 13, 305. A solution containing 1.44 micromoles ((imol) DTAF in 361 microliters {[ il)

of borate buffer, is added to 2.4 ml of 0.014 mM antibody and allowed to react for 1 hour at 25°C. The reaction mixture is chromatographed on a 1 x 25 cm "Biogel P-6DG" column with 0.1M sodium phosphate buffer, pH 7.0, 0.1M NaCl as eluent. The absorbances at 280 nm of . 1 ml fractions are checked

and those comprising the first peak are collected. The DTAF/protein molar ratio is determined by the method of The and Feltkamp (1970) Immunology 18, 865.

Free thiol groups are introduced into the antibody by reaction with N-succinimidyl 3-(2-pyridyldithio)propionate and subsequent mild reduction with dithiothreitol /Carlson et al., (1978) Biochem. J. 173, 723/.

B. A glucose oxidase antibody conjugate is prepared as follows. Maleimide groups are introduced into glucose oxidase (available from Miles Scientific, Naperville, IL) by reaction with N-hydroxysuccinic acid imide ester of N-(4-carboxy-cyclohexylmethyl)-maleimide (SMCC) as described by /Yoshi-toake, et al., ( 1979)0 Eur. J. Biochem. 101, 395 (1979)_/.

This glucose oxidase immediately combines with it

twice the molar amount of the thiol-derivatized

the antibody described above. The reaction conditions and purification procedures according to Yoshitake et al. is followed. The enzyme content in the purified conjugate is determined by measurement

of the enzyme activity and the antibody content is determined by measuring the DTAF content as described by The and Feltkamp, supra.

C. Horseradish peroxidase is reacted with SMCC and coupled

to the thiol-derivatized antibody by the method of Ishikawa et al., (1983) J. Immunoassay 4:209. D. A 565 base pair fragment from a gene for the 16th ribosomal RNA from E.coli is cloned between the Hind III positions of a pBR322 vector /Brosius (1978) Proe. Night'. Acad. Pollock. 75:4801/. The fragment is cleaved by digestion with Hind III restriction endonuclease.

E. A urine sample containing more than 10^ bacteria

per ml, diluted to three times the volume of an enriched culture broth and incubated at 35°C until the cell population reaches approx. 10 9 organisms per ml. Parts of the culture are transferred to a series of tubes, so that each tube contains 10"<*> to 10 9 bacteria. The tubes are centrifuged at 10,000 x g for 10 minutes and the supernatants are removed. The cells are lysed in 20 µl per tube of 10 milligrams of egg white lysozyme ( Sigma CHemical Co., St. Louis, MO) in 10 mM Tris-hydrochloride buffer, pH

8.0, 0.1M NaCl and 5mM ethylenediaminetetraacetic acid. 15

minutes later, 80 µl of a solution composed of 60% formamide and 40% 0.16 M sodium phosphate buffer, pH 6.5, is added

1.44 M NaCl, and 0.1% (w/v) sodium dodecyl sulfate).

The DNA probe described in section D above, in 0.2 M sodium phosphate buffer, pH 6.5, is denatured in a boiling water bath for 5 minutes and cooled on ice. 20 microliter samples (80 ng DNA) are added to each tube and incubated at 55°C

for 18 hours. Then 500 µl of peroxidase-labeled antibody and glucose oxidase-labeled antibody are added to each

tube that gets so at room temperature for 2 hours. The concentration of these enzyme conjugates is optimized in initial experiments, so that optimal signal-to-background ratios are achieved.

The following substrate reagent is prepared and used within

3 hours: 0.IM sodium phosphate, pH 6.5, 0.1% bovine serum albumin, 20 mM 3,5-dichloro-2-hydroxybenzenesulfonate, 0.IM glucose, 0.01 unit catalase/ml and 0.2 mM 4- aminoantipyrine. 1 ml of substrate reagent is added to each tube and incubated at 37°C for 30 minutes. The absorbances at 510 nm are recorded. When the number of bacteria per tube increases, the amount of ribosomal RNA"DNA hybrid will increase, and as a result, the absorbance will decrease.

Example II.

Hybridization assay for the detection of bacterial ribosomal RNA using a biotinylated DNA probe.

A. Biotin-II-dUTP and streptavidin-horseradish peroxidase conjugate are available from Enzo Biochem., Inc., New York,

NEW.

B. The 565 base pair probe for ribosomal RNA is labeled with biotin-11-dUTP by the method of Langer et al., (1981)

Pro. Nat., Acad. Sci., 78:6633.

C. Bacteria are processed as described in example I, part

E above. The lysates are combined with 80 µl of a solution consisting of 60% formamide and 40% 0.16M sodium phosphate buffer,

pH 6.5, 1.44M NaCl and 0.1% (w/v) sodium dodecyl sulfate. The biotinylated DNA probe is denatured in a boiling water bath for 5 minutes and then cooled on ice. The probe is in 0.2M sodium phosphate buffer, pH 6.5, and 20 µl aliquots containing 80ng probe are added to each extract and incubated at 65°C for 18 hours. 500 ul of peroxidase is then added

labeled streptavidin and glucose oxidase labeled antibody against RNA"DNA hybrid to each tube which is allowed to stand at room temperature for 2 hours. The concentrations of these enzyme conjugates are optimized in initial experiments so that maximal signal-to-background ratios are achieved.

The enzyme reactions are initiated by the addition of 1 ml of the substrate reagent described in example I, section D. These reactions are allowed to proceed at 37°C for 30 minutes and then the absorbances are recorded at 510 nm. The absorbances will increase when the number of bacteria added per pipe increases.

Example III.

Hybridization assay for bacterial ribosomal RNA using a probe intercalated with ethidium bromide.

A. Calve thymus DNA (Sigma Chemical Co.) is treated with

pronase to degrade protein contaminants and then dissolved in ethanol. DNA is dissolved in 20 mM Tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl.

A photoreactive derivative of ethidium bromide, 8-azidoethidium, is prepared by the method of Graves et al., (1977) Biochim. Biophys. Acta, 479:98. A solution containing approx. 1 mM base pair of DNA and 0.5 mM 8-azidoethidium. This solution is stirred in a reaction

glass vessels located in a water bath maintained at 20-30°C, and photolyzed 10-20 cm from a 150 watt spotlight for 60 minutes. After the photolysis, non-covalently bound ethidium derivative is removed by extractions with n-butanol saturated with water. The amount of ethidium linked to DNA is estimated by use

3—1—1

of extinction coefficients of E4 .ny un<=>4x10 M cm for photolysed ethidium azide, the relation between A260°^A490

for photolysed ethidium bound to DNA /& 2eo = /A4<gg><=>(<A>49g x 3.4)-0.01l7/og & 260~l'32xlO^M ^ cm 1 for the concentration of DNA base pairs for a given DNA being labeled.

B. Preparation of methylated thyroglobulin.

100 mg of bovine thyroglobulin (Sigma) is combined with 10 ml of anhydrous methanol and 400 µl of 2.55 M HCl in methanol. This mixture is stirred on a rotary mixer at room temperature for 15 days. The precipitate is collected by centrifugation and washed

2 times with methanol and 2 times with ethanol. It is then dried under vacuum overnight. It is achieved approx. 82

mg dry powder.

C. An immunogen is prepared by combining the DNA-ethidium complex with methylated thyroglobulin as indicated by Stoller (1980) Methods in Enzymology 70:70.

20-50 micrograms of the immunogen in Freund's adjuvant is injected per mice and booster injections are given weekly starting 2 weeks after the initial immunization. Antibody titers are measured using an ELISA procedure and hybridomas are prepared and examined for anti-

substance by standard procedures /Galfre and Milstein, (1981)

Meth. Enzymol. 73:1; Poirier, et al., (1982) PROc. Nati.

Acad. Sei., 79:6443/. Selection procedures were designed

for antibodies that bind the DNA-ethidium complex with association coefficients of 10"^ or greater, but which do not bind to single- or double-stranded DNA or the ethidium residue alone.

Antibody is isolated from peritoneal fluid such as the tear in the example

I, part A, for antibody to RNA"DNA hybrid. This antibody is also readily labeled with DTAF and coupled to glucose oxidase.

D. The 565 base pair fragment from the pKK115 plasmid is cloned

into the Hind III position on M13 mp9 /Brass and Vieria,

(1982) Gene 19, 269; commercially available from New Eng-

land Biolabs, Beverly, MA/. The fragment is inserted in two orientations and a clone with the virion DNA sequence complementary to the ribosomal RNA is selected for further work.

The modified M13 mp9 is propagated in E.coli JM103 /Alac, pro)thi, strA, supE, endA, sbcB15, hsdR4, F'traD36, proAB, laclq, ZAM15/ available from Bethesda Research Laboratories, Gaitherburg, MD. The phage is isolated from the culture broth by precipitation with polyethylene glycol and the virion DNA is purified by phenol/chloroform extraction /Maniatis et al., (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor/.

An oligonucleotide primer (CACAATTCCACACAAC; available

from New England Biolabs) is complementary to the M13 mp9 vector on the 5'-0H side of the inserted ribosomal RNA probe.

This primer is used to initiate the replication of

the M13 sequence using E.coli DNA polymerase (Klenow fragment) in the presence of limited amounts of deoxynucleotide triphosphates. Replication is limited to such an extent that the inserted ribosomal RNA probe remains single-stranded and available for hybridization /Hu and Messing (1982)

Gene 17:271/.

This modified M 13 probe is mixed with 8-azidoethidium, photolyzed and purified as described in part A above.

E. Bacteria are prepared and lysed as described in Example I, Part A. The lysates are mixed with 80 µl of the formamide hybridization solution described in Example I, Part E. 20 microliters of the ethidium probe complex (100 ng DNA) (part

D) is added to the extracts and incubated at 65°C for 18 hours, and then 500 ul of peroxidase-labeled antibody to RNA"DNA and glucose oxidase-labeled antibody are added

to the ethidium-DNA complex to each tube. The mixtures are allowed to stand at room temperature for 2 hours.

At the end of this incubation, add 1.0 ml

of the substrate reagent, Example I, Part E, and incubate at 37°C for 30 minutes. The absorbance is then recorded at 510 nm. When the number of bacteria per tube is increased, the absorbance will decrease.

Example IV.

Hybridization assays for cytomegalovirus controlled with a fluorescent-cloning pair.

A. A 1500 base pair fragment of the cytomegalovirus genome is cloned into the M13 mp 8 vector. A complementary copy of the inserted fragment is synthesized in vitro with DNA polymerase in the presence of a biotinylated nucleotide triphosphate so that a biotinylated probe is provided. The probe is further modified by covalent incorporation of ethidium residues as intercalation complexes.

An EcoRI degradation element of cytomegalovirus DNA is prepared and the fragments are cloned into the plasmid pACYC184 /Ta,asjorp et al., (1982) J. Virology 42:547/. The plasmids are propagated in E.coli strain HB101 Ree A and the EcoRI e fragment described by Tamashiro is used to prepare the probe. The fragment is removed from the plasmid by digestion with the EcoRI restriction enzyme and cloned into the EcoRI position on the Ml3mp8 vector (New England Biolabs) which is propagated in the E.coli K12JM101 host. The virus is isolated from the culture liquid by precipitation with polyethylene glycol and the DNA is purified by phenol/chloroform extraction.

The DNA is recombined with a 17 base primer with the sequence GTAAAACGACGGCCAGT (New England Biolabs) which is complementary to a region of the Ml3mp8 sequence near the 3'-OH end of EcoRI

e the deposit. DNA in 20 mM Tris-hydrochloride buffer, pH 8.0, containing 10 mM MgCl 2 is combined with a molar excess of the primer and incubated at 55°C for 45 minutes. /Bankier and Barrell (1983) Techniques in Nucleic Acid Biochemistry, Elsevier, Ireland/. The reaction mixture is made 15 mM with regard to dATP, dCTP, dGTP and Bio-ll-dUTP (available from Enzo Biochem) and finally the Klenow fragment of DNA polymerase I is added. This reaction is incubated at 25°C for a period of time determined by initial experiments.

In these experiments, samples are taken from the reaction mixture at different times and electrophoresed in alkaline agarose gel /Maniatis et al., supra/. The goal is to find reaction conditions that give biotin-labeled probes that extend through the EcoRI e insert. This method will provide biotin-labeled DNA of somewhat variable length; however, extension of the labeled DNA is beyond EcoRI

e the deposit into M13 is acceptable for present purposes.

The next step is the covalent coupling of ethidium residues to the biotin-labeled DNA as intercalation complexes. This is achieved by photolysis of DNA with 8-azidoethidium prepared by the method according to Graves et al., supra. Biotin-labeled DNA is extracted with phenol/chloroform and precipitated with ethanol. It is dissolved in 20 mM Tris-hydrochloride buffer,

pH 8.0, 0.2 M NaCl, so that it gives approx. 50 µg DNA/ml and made 0.5 mM with respect to 8-azidoethidium. The mixture is photolyzed for 1 hour 10-20 cm. from a 150 watt floodlight. The mixture is stirred during this time in a glass reaction vessel which is placed in a glass water bath. The bath maintains a reaction temperature of 20-30°C.

The non-covalently bound 8-azidoethidium is then removed

and the photolysis by-products by means of successive extractions with n-butanol saturated with water. DNA is precipitated with ethanol and dissolved in the tris buffer. The covalently bound ethidium is measured spectrophotometrically as described in example III,

part A.

8-azidoethidium binds covalently to DNA almost exclusively in the double-stranded region where intercalation complexes can form. Measure is to covalently link an ethidium residue per 10-50 base pairs. Since the photolytic reaction proceeds almost completely within 1 hour, the incorporation can be reduced by reducing the reaction time or by reducing the concentration of 8-azidoethidium. Incorporin gene can be increased by repeating the photolysis with fresh 8-azidoethidium.

The final step is separation of the biotin-labelled/ethidium modified probe from the M13mp8 template. The labeled probe is substantially smaller than the Ml3mp8 template and can be isolated by alkaline agarose gel electrophoresis, /Maniatis et al., supra/. The part containing the probe is cut from the piece of agarose gel and recovered by electro-elution as described by Maniatis.

B. Fluorescein-labeled antibody to ethidium-DNA intercalation complex.

Monoclonal antibody to ethidium-DNA complex described

in Example III, Part B above, dialyze into 0.1M sodium borate buffer, pH 9.0, and 0.5 ml containing 5 mg of antibody, combine with 0.5 ml of 10 mM 5 (4,6-dichlorotriazine -2-yl)-aminofluorescein (Molecular Probes, In., Junction City, OR) in the borate buffer. The mixture is allowed to stand for 7 hours

at room temperature and then chromatographed on a 1x26 cm column of "Biogel P6DG" resin in 0.1 M tris-hydrochloride buffer,

pH 8.0. The absorbances at 280 nm of 1.0 ml fractions are measured, and the fractions from the first eluted peak are pooled. The fluorescein/protein ratio is measured spectrophotometrically

by the method according to The and Feltkamp (1970) Immunology 18:865. C. Preparation of streptavidin labeled with 4,5-dimethoxy-6-dicarboxyfluorescein.

The N-hydroxysuccinimide ester of 4', 5'-dimethoxy-6-carboxyfluorescein is synthesized by the methods of Khanna and Ullman (1980) Anal. Biochem. 108:156. (This synthesis gives in practice a mixture of the 5 and 6-carboxyfluorescein isomers). Streptavidin isolated from Streptomyces avidini /Hoffman et al., (1980) Proe. Nati. Acad. Pollock. 77:4666/ is labeled with the N-hydroxysuccinic acid imide ester of 4',5<1->dimethoxy-6-carboxyfluorescein by the method described by Khanna and Ullman for labeling immunoglobulins.

D. Hybridization assay for cytomegalovirus in urine.

In this method, centrifugation is used to isolate the virus from the urine and the viral DNA is hybridized in solution with the biotin-labelled/ethidium-modified probe. Then, the labeled avidin and the antibody to the ethidium-DNA intercalation complex are allowed to bind to the hybrids

that is formed and fluorescence is used to estimate the amount of hybrid present.

Cellular and particulate materials are removed from the urine by centrifugation in a "Sorvall GLC-3" centrifuge at 3000 rpm for 5 minutes. The supernatant is placed in a poly-alloyed ultracentrifuge tube and run at 25,000 rpm in a "Beckman Ti50" rotor for 75 minutes. The pellet is dissolved in 0.1 M NaOH and incubated at 37°C for 30 minutes.

150 microliters of 0.2 M sodium phosphate buffer, pH 6.0, containing 1.8 M NaCl, 0.1% sodium dodecyl sulfate (w/v)

and 1 mM EDTA is added. Then 20 µl of the biotin-labelled/ethidium-modified probe (50 mg) is added and the hybridization mixture is incubated at 65°C for 10 hours. After the hybridization reaction, 650 µl of 0.1 M tris-hydrochloride buffer, pH 8.2, containing the labeled avidin and antibody to the ethidium-DNA intercalation complex is added. The concentration of the labeled proteins in this reagent is optimized in initial experiments to maximize the ratio of fluorescence shutoff to background. The protein binding reactions are allowed to take place at room temperature for 1 hour.

The fluorescence of the reaction mixture is then recorded using 495 nm light for excitation and 519 nm for emission. A reference assay is run in parallel with a urine sample known to be free of cytomegalovirus, and the fluorescence signal obtained by this reference assay will be higher than the signals obtained for samples containing the virus, consequently the fluorescence signal will decrease as the virus concentration increases.

Claims (15)

1. Method for detecting a particular polynucleotide sequence in a sample that includes single-stranded nucleic acids, where the method includes bringing the sample into contact with a nucleic acid probe that contains at least one single-stranded base sequence that is essentially complementary to the sequence to be detected, and determination of the resulting hybrids, characterized in that the hybrids are formed such that they have binding positions for two specific binding reagents, any hybrid formed is contacted with the two specific binding reagents, one of which includes a first label and the other includes a second label, the interaction between the first and second labels produces a detectable response that is measured differently when the two labeled reagents are both bound to the same hybrid compared to the case when they are not so bound, and the detectable response is measured as a function of of the presence of the sequence to be detected in the sample. 2. Method according to claim 1, characterized in that the first label participates in a first chemical reaction which forms a diffusible product which is a participant in a second chemical reaction with a second label, so that a product is formed which provides the detectable response, the first and second labels are preferably catalysts for the first and second chemical reactions, respectively. 3. Method according to claim 1, characterized in that the first and second marks participate in an energy transfer interaction. 4. Method according to claim 1, characterized in that one of the specifically binding reagents is an antibody reagent. 5. Method according to claim 4, characterized in that the antibody reagent is a reagent which is selective for binding to DNA"RNA hybrids where either the probe or the sequence to be detected is DNA and the other is RNA, or one which is selective binding to RNA"RNA hybrids if both the sample and the sequence to be detected are RNA. 6. Method according to claim 4, characterized in that the antibody reagent is one that selectively binds to intercalation complexes, where the duplex formed in the analysis includes a nucleic acid intercalator bound thereto in the form of intercalation complexes, preferably the intercalator is chemically bound to the probe in its single-stranded complementary region , whereby upon hybridization with the sequence to be detected, intercalation complexes are formed in the resulting hybrid. 7. Method according to either claim 5 or 6, characterized in that (i) the second specifically binding reagent includes the same antibody reagent, (ii) the nucleic acid probe includes a specifically binding ligand part, and the second specifically binding reagent is a binding partner for a such ligand moiety, or (iii) the nucleic acid probe includes a double-stranded moiety having a binding site for a protein and the other specifically binding reagent is such a protein. 8. Method according to claim 1, characterized in that the sample includes a biological sample that has been exposed to conditions that release and denature nucleic acids that are present in the sample. 9. Reagent system for detecting a particular polynucleotide sequence in a sample that includes a nucleic acid probe that contains at least one single-stranded base sequence that is essentially complementary to the sequence to be detected, characterized in that it additionally includes first and second specifically binding reagents capable of to bind to the hybrids formed between the sequence to be detected in the sample and the probe, such binding reagents comprising first and second labels, respectively, which interact with each other and provide a detectable response that is measurably different when the two labeled reagents are both bound to the same the hybrid, compared to the case that they are not bound in this way. 10. Reagent system according to claim 9, characterized in that the first label participates in a first chemical reaction that produces a diffusible product that is a participant in a second chemical reaction with the second label, so that a product is formed that provides the detectable response, the first and second labels are preferably catalysts for the first and second chemical reactions, respectively. 11. Reagent system according to claim 9, characterized in that the first and second label participate in an energy transfer interaction. 12. Reagent system according to claim 9, characterized in that one of the specifically binding reagents is an antibody reagent. 13. Reagent system according to claim 12, characterized in that the antibody reagent is one which is selective binding to DNA"RNA hybrids where either the probe or the sequence to be detected is DNA and the other is RNA, or one which is selective binding to RNA'RNA hybrids where both the probe and the sequence to be detected are RNA. 14. Reagent system according to claim 12, characterized in that the antibody reagent is one that selectively binds to intercalation complexes, where the duplexes formed in the analysis include a nucleic acid intercalator bound thereto, in the form of intercalation complexes, preferably the intercalator is chemically bound to the probe in its single-chain complementary region , whereby upon hybridization with the sequence to be detected, intercalation complexes are formed in the resulting hybrid. 15. Reagent system according to claim 13 or 14, characterized in that (i) the second specifically binding reagent includes the same antibody reagent, (ii) the nucleic acid probe includes a specifically binding ligand part, and the second specifically binding reagent is a binding partner for such ligand moiety, or (iii) the nucleic acid probe includes a double-stranded moiety having a binding site for a protein, and the second specific binding reagent is such a protein.