HK1169452A - A set of oligonucleotide probes as well as methods and uses related thereto - Google Patents

Description

Oligonucleotide probe set and methods and uses related thereto

In a first aspect, the invention relates to a set of oligonucleotide probes comprising at least 100 different single-stranded oligonucleotide probes for a genomic target sequence of interest, wherein each individual oligonucleotide comprises at least one label. In a further aspect, the invention relates to the use of said set of oligonucleotide probes for detecting a genomic target sequence. In another aspect, the invention relates to a method of detecting a genomic target sequence of interest, said method comprising the steps of incubating a set of oligonucleotide probes according to the invention and a sample under conditions conducive to binding of the set of oligonucleotide probes to the genomic target sequence of interest, and detecting binding of the oligonucleotide probes to the genomic target sequence. In a further aspect, the present invention provides a method of generating a set of oligonucleotide probes directed against a genomic target sequence of interest, said method comprising the steps of designing a set of oligonucleotide probes complementary to at least 100 different regions of the genomic target sequence of interest and synthesizing said set of oligonucleotide probes. In a still further aspect, the invention relates to a kit comprising a set of oligonucleotide probes according to the invention, in addition comprising further components selected from the group consisting of: deparaffinization reagents, pretreatment reagents, washing reagents, detection reagents and product sheets.

In Situ Hybridization (ISH) has become an indispensable tool in many fields of research and clinical diagnostics. In Situ Hybridization (ISH) is a unique technique in which molecular biology and histochemical techniques are combined to study gene expression in tissue sections or cytology, as it allows the detection and localization of both DNA and RNA in specific cells. ISH analysis was first described in 1969 and involves a hybridization reaction between a labeled nucleotide probe and a complementary DNA or RNA, the hybrids of which can be detected by a variety of procedures depending on the nature of the label included. The introduction of non-radioactive probe labeling and detection systems in the late 70 s made in situ hybridization analysis possible as a molecular diagnostic tool in diagnostic pathology laboratories. ISH localizes gene sequences in situ, allowing visualization of the products of gene expression while maintaining cellular integrity within heterogeneous tissues. The main advantages of the ISH method are its specificity for individual targets in heterogeneous tissues or cell populations, and its sensitivity in detecting low copy gene expression in cell or chromosome mapping. ISH consists of multiple steps, including probe preparation and labeling, tissue preparation, hybridization, and signal detection (for a review see, e.g., Jin et al, 2001, Morphology Methods: Cell and Molecular Biology Techniques: 27-46).

Southern and slot blots were the first gene-based HER-2 detection method used in breast cancer specimens. FISH (fluorescence in situ hybridization) technology, which is morphologically driven and can be automated, like IHC, has the advantage of a more objective scoring system and the presence of a built-in internal control consisting of the two HER-2 gene signals present in all non-neoplastic cells in the sample. Disadvantages of FISH testing include the high cost of each test, the long time required for slide scoring, the need for fluorescence microscopy, the inability to store slides for storage and review, and sometimes in identifying invasive tumor cells. To date, the FDA approved two versions of ISH analysis: ventana Inform^TMTest for measuring only HER-2 gene copies, and Abbott-Vysis Pathvysis^TMTests, which include chromosome 17 probing in a two-color format, both of which are described as being highly correlated (for review see, e.g., Rosset et al, 2004, Molecular and Cellular Proteomics 3.4: 379-.

Chromogenic in situ hybridization methods (also known as CISH techniques) are characterized by the advantages of both immunohistochemistry (i.e., conventional microscopy, low cost) and FISH (i.e., built-in internal controls, objective scoring, more efficient DNA targeting). For example, both FISH and CISH methods were used to compare 31 cases of invasive breast cancer in tests conducted in laboratories at two institutions, and the same results were obtained in 26 cases (84%) (Guptaet al, 2003, am.j.clin.pathol., 119: 381-. CISH and IHC detection methods can be combined to provide simultaneous assessment of gene copy number and protein expression, but such methods have not been adopted in clinical practice.

Three principle types of probes can be used for in situ hybridization, including (i) double-stranded complementary DNA probes, (ii) single-stranded antisense RNA probes, and (iii) synthetic oligonucleotide probes. As with other hybridization techniques, probe design, synthesis and labeling play a critical role in the success of in situ hybridization in terms of specificity and sensitivity. Standard labelling procedures generally involve the use of enzymatic methods. Double-stranded DNA probes can be labeled by enzymatic nick translation or random primer methods, and antisense RNA probes are usually synthetic and labeled by in vitro transcription methods. However, these methods result in the generation of non-defined probes, where the position and number of markers is not defined and unknown to the physician. In addition, enzyme labeling requires more skilled personnel to perform such labeling because the amount of label incorporated depends on a range of variables, including, for example, enzyme activity and/or the amount of label present. Finally, each label may have different results for the exact location and amount of incorporated label, since the labeling reaction is the result of a random process.

However, oligonucleotide probes are typically labeled by either 5 'or 3' end labeling methods, including, for example, by "tailing" of the probe with incorporation of relatively small amounts of labeled nucleotides. The sensitivity of end-labeled probes is rather low due to the limited number of labels per probe.

Oligonucleotide probes used for in situ hybridization are mostly short in length and generally less sensitive than longer cDNA or antisense RNA probes. Thus, to date, oligonucleotide probes have been mostly used to detect abundant and/or repetitive gene targets, or to map the expression of specific gene families in the brain (see, e.g., Wisden and Morris, 2002, International Review of Neurobiology, vol.47, Chapter 1). Gene targets analyzed by in situ hybridization procedures using oligonucleotide probes further include, for example, abundant mRNA transcripts such as β -actin (Perlette and Tan, 2001, AnalChem 73: 5544-.

However, the use of oligonucleotide probes for the detection and/or analysis of genes present only to a low extent, including for example low abundance tumor marker genes, by routine and high throughput application of hybridization methods would be of particular interest in modern medicine, diagnostics and cancer therapy, as oligonucleotide probes can be readily synthesized in high amounts and at low cost. Thus, there is a need for sensitive detection methods that utilize oligonucleotide probes.

It is therefore an object of the present invention to provide alternative methods for detecting genes, in particular low abundance target sequences requiring sensitive detection methods.

In the context of the present invention, it has been found that a method using a "cocktail" (cocktail) of at least 100 different single-stranded oligonucleotide probes complementary to different regions of a target molecule is particularly suitable for detecting low-abundance genes. In particular, it has been found that such mixtures are particularly suitable for the detection of genomic target sequences of interest at reduced time intervals (see FIG. 1), require reduced amounts of probes (see FIG. 2) and have higher specificity (see FIGS. 1-3) compared to detection methods known and described in the art (see, e.g., Weiss et al, 1990, American Journal of Pathology, Vol.137, No. 4: 979-. Thus, it is apparent that such methods are superior to those known and described in the art in that they are faster, cheaper, more sensitive and/or more predictable.

Thus, in a first invention, the invention provides a set of oligonucleotide probes comprising at least 100 different single-stranded oligonucleotide probes for a genomic target sequence of interest, wherein each individual oligonucleotide probe comprises at least one label.

Single-stranded oligonucleotide probes are particularly beneficial for detecting genomic sequences of interest because (i) no heating step is required to ensure disintegration of the secondary structure, and (ii) re-annealing of the complementary strand of the probe is omitted.

The term "oligonucleotide probe" as used herein generally refers to any type of nucleotide molecule that is synthesized to match a nucleotide sequence of interest, which can be used to detect, analyze, and/or visualize the nucleotide sequence at the molecular level. An oligonucleotide probe according to the present invention generally refers to a molecule comprising (i) several nucleotides, generally at least 10, more preferably at least 15, and most preferably at least 20 nucleotides, and (ii) at least one label. Optionally, the oligonucleotide probe may further comprise any suitable non-nucleotidic unit and/or a linking reagent suitable for incorporation of a label. It will be apparent to the skilled person that the oligonucleotide probe has a length suitable to provide the required specificity. In general, the probe may be a DNA oligonucleotide probe or an RNA oligonucleotide probe, preferably a DNA oligonucleotide probe. Accordingly, nucleotides include all types of structures consisting of nucleobases (i.e., nitrogenous bases), five-carbon sugars, which can be ribose, 2' -deoxyribose, or any derivative thereof, and phosphate groups. Nucleobases and sugars constitute what are called nucleosides. The phosphate group may form a bond with a 2, 3 or 5-carbon, particularly the 3 and 5 carbons of the sugar. In the context of the present invention, the term "nucleotide" likewise refers to a ribonucleotide or a deoxyribonucleotide. Ribonucleotides contain ribose as the sugar moiety, while deoxyribonucleotides contain deoxyribose as the sugar moiety. The nucleotide may contain a purine or pyrimidine base. Thus, the oligonucleotide probe of the present invention may be composed of ribonucleotides or deoxyribonucleotides, or any combination thereof, and may further include one or more modified nucleotides. Optionally, the oligonucleotide probe may further comprise only modified nucleotides. The ribose-or deoxy-forms of modified nucleotides may, for example, include, but are not limited to, 5-propynyl-uridine, 5-propynyl-cytosine nucleoside, 5-methyl-cytosine nucleoside, 2-amino-adenosine, 4-thiouridine, 5-iodouridine, N6-methyl-adenosine, 5-fluorouridine, inosine, 7-propynyl-8-aza-7-deaza-purine and 7-halo-8-aza-7-deaza-purine nucleosides. The oligonucleotide probe of the present invention may further comprise a stem modification, such as, for example, 2 '-O-methyl (2' -OMe) RNA, 2 '-fluoro (2' -F) DNA, Peptide Nucleic Acid (PNA), or Locked Nucleic Acid (LNA). 2' -OMe RNA is a particularly suitable stem modification because it hybridizes more strongly than DNA and, unlike natural RNA, is extremely stable to RNases and DNases. LNA is a class of conformationally constrained oligonucleotide analogues in which ribonucleosides are linked to methylene groups between 2 '-O and 4' -C. It is similar to natural nucleic acids in base pair and duplex formation, but the LNA duplex is more thermostable than the corresponding DNA or RNA duplex (Silverman and Kool, 2007, advanced sin Clinical Chemistry, Vol.43: 79-115). Optionally, the oligonucleotide probes of the invention may further comprise one or more modifications on the phosphate backbone, e.g., phosphorothioate or methylphosphonate, which improve stability to nucleases.

A set of oligonucleotide probes according to the invention refers to a pool of at least 100 different oligonucleotides. These oligonucleotides may differ from each other in one or more nucleotide positions, or may differ from each other in their complete sequence. Preferably, a set of oligonucleotide probes of the invention comprises more than 100 different oligonucleotides, in particular at least 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 different oligonucleotides. More preferably, the set of oligonucleotide probes comprises at least 1000 different oligonucleotide probes, in particular at least 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 different oligonucleotide probes.

Thus, in a preferred embodiment, a set of oligonucleotide probes according to the invention comprises at least 200, 300 or 400 different oligonucleotide probes, in particular at least 500 different oligonucleotide probes, more in particular at least 1000 different oligonucleotide probes. As the number of different oligonucleotide probes increases, the sensitivity and specificity of the detection method increases (see FIG. 3).

Furthermore, the set of oligonucleotide probes according to the invention is further characterized in that each oligonucleotide probe has a defined length. Such length is comprised of a defined number of nucleotides, optionally further comprising a defined number of non-nucleotide linking reagents. The generation of oligonucleotide probes having a defined length provides advantages over methods known in the art that employ a fragmentation step, thus resulting in the generation of oligonucleotide probes of random length distribution. Preferably, the set of oligonucleotide probes of the invention is characterized in that each oligonucleotide probe has a defined length of 20 to 200 nucleotides, or 40 to 175 nucleotides, or 60 to 150 nucleotides, or 80 to 120 nucleotides. However, it is obvious to the skilled person that the above upper and lower limits may also be combined to achieve different ranges. Thus, the oligonucleotide probe may also have a length of 20 to 150, 60 to 120, or 80 to 150, or 30 to 180. The set of oligonucleotide probes of the invention may comprise oligonucleotide probes of all the same length or, if deemed appropriate, may comprise oligonucleotide probes of different lengths.

Thus, in a preferred embodiment, the set of oligonucleotide probes of the invention is characterized in that each oligonucleotide probe has a length of 20 to 200 nucleotides, preferably 40 to 175 nucleotides, more preferably 60 to 150 nucleotides, in particular 80 to 120 nucleotides.

Oligonucleotide probes having a length of about 80 to 120 nucleotides have been found to be particularly suitable for detecting genomic target sequences according to the invention.

As a prerequisite to obtaining the results described in the context of the present invention, the set of oligonucleotide probes must further comprise a minimum total amount of incorporated labels. That is, the shorter the length of the oligonucleotide probe, the more labels per oligonucleotide must be incorporated, attached, conjugated or linked to each individual molecule, or more oligonucleotide probes should be used to provide a set of oligonucleotides suitable for detecting genomic target sequences of interest under the conditions described therein. To control the incorporation of labels, the oligonucleotide probes of the invention are preferably generated by chemical synthesis. The advantages of chemical synthesis over the use of enzymatic methods are a rapid and cost-effective probe generation process, and control of the precise fragment length, sequence, number and location of labels. For example, in the context of the present invention, it was found to be particularly advantageous if the oligonucleotide probe comprises at least five labels in the presence of about 80 to 90 nucleotides. Preferably, the oligonucleotide probe comprises at least eight or more labels in the presence of about 80 to 90 nucleotides. Oligonucleotides according to the invention comprising a defined number of incorporated labels are illustrated, for example, in FIG. 5.

Thus, in another preferred embodiment, the set of oligonucleotide probes is characterized in that each oligonucleotide probe comprises at least two or three labels, in particular at least five labels, more in particular at least 10 labels, most in particular at least 15 labels.

As found in the context of the present invention, oligonucleotide probes show an increased sensitivity with increasing incorporation of labels. However, care must be taken that the presence of too many labels does not affect successful hybridization and therefore functionality. Thus, the presence of 5 to 15 labels per oligonucleotide (depending on the length of the individual molecule) was found to establish the best results.

Most preferably, the set of oligonucleotide probes of the invention is characterized in that (i) each oligonucleotide probe has a length of 20 to 200 nucleotides, preferably 40 to 175 nucleotides, more preferably 60 to 150 nucleotides, especially 80 to 150 nucleotides, and (ii) each oligonucleotide probe comprises at least two or three labels, especially at least five labels, more especially at least 10 labels, most especially at least 15 labels.

In particular, each oligonucleotide probe comprises at least five labels and has a length of 20 to 200 nucleotides, preferably 40 to 175 nucleotides, more preferably 60 to 150 nucleotides, especially 80 to 150 nucleotides.

More preferably, each oligonucleotide probe comprises at least 10 labels and has a length of 20 to 200 nucleotides, preferably 40 to 175 nucleotides, more preferably 60 to 150 nucleotides, especially 80 to 150 nucleotides.

More preferably, each oligonucleotide probe comprises at least 15 labels and has a length of 20 to 200 nucleotides, preferably 40 to 175 nucleotides, more preferably 60 to 150 nucleotides, especially 80 to 150 nucleotides.

Alternatively, each oligonucleotide probe has a length of 60 to 150 nucleotides and comprises at least two or three labels, particularly at least five labels, more particularly at least 10 labels, most particularly at least 15 labels.

More preferably, each oligonucleotide probe has a length of 80 to 150 nucleotides and comprises at least two or three labels, in particular at least five labels, more in particular at least 10 labels, most in particular at least 15 labels.

The term "directed to" in the context of the present invention generally means that the oligonucleotide probes of the present invention can bind to a target sequence of interest by means of complementary base pairs. Complementary base pairs are formed between two nucleotide molecules (optionally including modifications) that are complementary to each other. In the context of the present invention, complementary base pairs formed between an oligonucleotide probe and a target sequence may include all types of canonical or atypical base pairs, including, but not limited to, Watson-Crick A-U, Watson-Crick A-T, Watson-Crick G-C, G-U Wobble base pairs, A-U and A-C reverse Hoogsteen base pairs, or purine-purine and pyrimidine-pyrimidine base pairs, e.g., cleaved G-A base pairs or G-A imino base pairs.

The term "genomic target sequence" as used herein generally refers to a specific region of the genome of a eukaryotic organism. In the context of the present invention, a genomic target sequence preferably refers to a defined region of the human genome. The haploid human genome consists of a total of more than three billion DNA base pairs, containing an estimated about 30,000 protein coding sequences. That is, only about 1.5% of the genome encodes proteins, while the remainder contains thousands of RNA genes, e.g., genes encoding trnas, ribosomal RNAs, micrornas, or other non-coding RNAs, regulatory sequences, introns, or repetitive sequences. Repetitive sequences of the human genome include, but are not limited to, centromere, telomere, tandem repeats, e.g., satellite DNA, mini-satellite DNA, microsatellite DNA, and interspersed repeats, such as SINE (short interspersed nuclear element) and LINE (long interspersed nuclear element). Thus, the genomic target sequence of the present invention may include, but is not limited to, the sequence of a particular gene or portion thereof, and may further include regulatory sequences, non-coding sequences, repetitive or non-repetitive sequences. Preferably, the genomic target sequence of the present invention refers to a defined locus comprising a specific gene.

Typically, genes are distributed irregularly across the genome of an organism, i.e., across different chromosomes. That is, a genomic target sequence of the invention may refer to a region of variable length, and/or to a region that is spread over different regions of one chromosome, and/or even spread over different chromosomes. In particular, a genomic target sequence may refer to one or more individual regions. Preferably, the genomic target sequence of the present invention refers to a locus, i.e., to a specific region in a chromosome where a gene is located. Genomic target sequences of the invention may include regions of variable size, including, but not limited to, regions having a size of up to 1 MB.

However, it is to be understood that the oligonucleotide probes are directed against different target sequences, i.e., against different regions within the genomic target sequence of the invention. That is, when a set of oligonucleotide probes comprising at least 100 different individual oligonucleotide probes collectively are directed against a genomic target sequence, which preferably comprises a specific locus, each individual oligonucleotide probe is directed against a specific subsequence located within this region.

The term "label" as used herein generally refers to any type of substance or reagent that can be incorporated into and/or attached to an oligonucleotide probe of the invention and which can be used to visualize, detect, analyze and/or quantify the oligonucleotide probe when it binds to its target sequence. Labels according to the present invention can include, but are not limited to, radioisotopes, such as,³⁵sulfur (35S),³²Phosphorus (32P),³³Phosphorus (33P),³H or¹⁴C, any fluorescent molecule or fluorophore that can be detected or visualized by means of fluorescence analysis, for example, fluorescein dyes, including, but not limited to, carboxyfluorescein (FAM), 6-carboxy-4 ', 5' -dichloro-2 '7' -dimethoxyfluorescein (JOE), Fluorescein Isothiocyanate (FITC), tetrachlorofluorescein (TET), and hexachlorofluorescein, rhodamine dyes, e.g., carboxy-X-Rhodamine (ROX), Texas Red, and Tetramethylrhodamine (TAMRA), cyanine dyes, e.g., pyraneCyanine dyes, DY548, Quasar 570 or Cy3, Cy5, Alexa 568, and the like. The choice of fluorescent marker is generally determined by its spectral properties and the availability of imaging equipment. Fluorescent markers are commercially available from various suppliers, including, for example, Invitrogen^TM(USA)。The marker of the invention may also be a hapten, such as Digoxigenin (DIG), biotin, or 2, 4-Dinitrophenyl (DNP). These markers and their detection strategies are well known to those skilled in the art and are described, for example, in Jin et al, 2001(Morphology Methods: Cell and molecular Biology Techniques, 27-48), Krick, 2002(Ann. Clin. biochem. 39: 114-12) or Grzybowski et al, 1993(Nucleic Acids Research, Vol.21, No. 8: 1705-1712).

Furthermore, labels of the invention may be attached to the oligonucleotide probe by chemical conjugation to a nucleotide or non-nucleotide linking reagent (i.e., direct labeling), or by chemical conjugation of a nucleotide or non-nucleotide linking reagent to a molecule that can bind to the label (i.e., indirect labeling). In indirect labeling, the molecule directly attached to the oligonucleotide probe is typically a hapten, such as 2, 4-Dinitrophenyl (DNP), Digoxigenin (DIG), or biotin. In direct labeling, the molecule attached to the oligonucleotide probe is typically a fluorescent dye. The detection strategies for different labels according to the present invention are described in further detail below.

The label of the invention may further be attached to the 5 '-end and/or the 3' -end of the oligonucleotide probe, or it may be incorporated internally into the oligonucleotide probe by a linkage to one or more modified nucleotides or phosphates. Alternatively, the labels of the present invention may also be incorporated into the oligonucleotide probes by linkage to one or more non-nucleotide linking reagents. Optionally, the label may be incorporated into the oligonucleotide probe during the 2-step process, for example, by introducing a reactive group such as an amine, azide, or alkyne group into the oligonucleotide probe, and coupling the label in a second step by amide bond formation or click chemistry (click chemistry). The term "click chemistry" as used herein generally refers to any bio-orthogonal (bioorthogonal) reaction that allows for the efficient formation of specific products within a highly complex chemical environment. In particular, it refers to a chemical reaction that allows the incorporation of one or more reactive tags on a biomolecule target and the subsequent high selectivity in tag derivatization within complex biological samples. The principles of click chemistry are known to those skilled in the art and are described, for example, in Best, M.D. (Biochemistry 2009, 48 (28): 6571-. Another way of introducing the labels of the invention is, for example, by coupling the label during the oxidation step of phosphoramidite based oligonucleotide synthesis, e.g. as described in WO 2007/059816.

The set of oligonucleotide probes of the invention may be designed such that each individual oligonucleotide is present in one or more copies, preferably in multiple copies. Preferably, the set of oligonucleotide probes is designed such that each individual oligonucleotide probe is present in equimolar amounts and/or at similar concentrations. Methods for calculating the concentration and/or molarity of nucleic acid molecules are standard knowledge of those skilled in the art. Furthermore, the set of oligonucleotide probes is preferably designed such that each copy of an individual oligonucleotide probe has the same sequence and contains one or more labels at the same position. Each of the individual oligonucleotide probes, present in single or multiple copies, constitutes a subset of the oligonucleotide probes.

Thus, in another preferred embodiment, the set of oligonucleotide probes of the invention comprises one or more subsets of oligonucleotide probes, wherein each subset consists of oligonucleotide probes having the same sequence with labels at the same position.

As found in the context of the present invention, a subset of oligonucleotide probes having the same sequence and having labels at the same positions is particularly advantageous, since their biochemical properties are known to the experimenter, as a result of which the most advantageous positions for incorporating labels can be selected, and furthermore, unfavorable positions, for example, positions that would cause an unfavorable quenching of the fluorescent signal, can be prevented.

The oligonucleotide probe of the present invention may be further characterized in that a label is inserted to a defined position during chemical synthesis of the oligonucleotide. That is, a defined number of labels can be incorporated into the molecule, by incorporating modified nucleotides at defined positions where the labels are to be attached, linked and/or conjugated, or by incorporating non-nucleotide linking reagents that serve as platforms for label attachment. The insertion of the label at a defined position also includes the manipulation of incorporating the label during the oxidation step of phosphoramidite-based oligonucleotide synthesis (see, e.g., WO 2007/059816). Preferably, the incorporation of the label is such that the label is uniformly distributed on the oligonucleotide probe. More preferably, the oligonucleotide probes are designed such that the labels are inserted into the molecule at mutually equidistant positions, in particular at mutually spaced distances of at least 5, 10, 15 or 20 or more nucleotides. Such a separation distance may be different from one subset of oligonucleotide probes to another, and may also depend on the total length of the individual oligonucleotide probes, i.e., the separation distance between individual labels may increase depending on the total length of the oligonucleotide probes.

Thus, in preferred embodiments, the oligonucleotide probes of the invention comprise labels at approximately equally spaced positions, in particular at least 5, 10, 15, or 20 nucleotides apart.

In another preferred embodiment, the oligonucleotide probe may comprise labels at predetermined positions, optionally at positions located at a predetermined distance from each other.

An advantage of locating the labels at a particular separation distance is that any side effects that may occur if the labels are too close to each other (e.g. interference and/or quenching if fluorescent labels are used) can be avoided. Furthermore, by controlling the incorporation of labels, it is further possible to avoid introducing too much label at certain positions that affect the efficiency of hybridization.

The term "predetermined" as used herein generally means that when designing a single oligonucleotide probe, the location at which a label is incorporated, attached, conjugated or ligated can be defined. That is, the term "predetermined" means that the labels are not randomly distributed and/or incorporated, for example, as in enzymatic labeling methods. One possibility to identify and/or determine the position of a predetermined marker is if the distribution of the marker within a molecule follows a certain pattern. Furthermore, the predetermined position may also be evident when analyzing the distribution of labels within one subset, for example, if each oligonucleotide probe of one or more subsets shows the same pattern of incorporated labels.

As detailed above, the label may be incorporated, attached, conjugated or linked directly or indirectly to a single oligonucleotide probe.

Thus, in a preferred embodiment, the set of oligonucleotide probes of the invention is characterized in that the label is attached directly or indirectly via a linker to the base, sugar, or phosphate moiety of the nucleotide.

By attaching labels to base, sugar or phosphate moieties, (i) possible damage and/or injury of complementary base pairs to the target sequence is minimized, and (ii) short oligonucleotide probes can be used to achieve efficient binding affinity.

For example, a 2, 4-Dinitrophenyl (DNP) label may be attached directly to the cytosine nucleoside at the N4 position. Alternatively, the nucleotides may be modified to allow and/or simplify the attachment of labels. That is, for example, pyransThe cyanine dye may be covalently linked to a 5-aminoalkyl-modified pyrimidine nucleotide. Direct attachment of the label to the probe preferably does not directly modify the site on the purine or pyrimidine base that normally involves hydrogen bonding interactions of complementary base pairing.

The term "linker" as referred to herein generally refers to any chemical structure that may be used to couple, conjugate, covalently bind or attach a tag to the nucleobase, sugar or phosphate portion of a nucleotide. Preferably, linkers according to the present invention may include, but are not limited to, flexible or rigid structures such as ethylene glycol linkers, hexaethylene glycol linkers, propargylamino linkers, aminoalkyl linkers, cyclohexyl linkers, aryl linkers, ethylene glycol alkynyl and ethoxyethylamino linkers, or alkylamine linkers. In general, the label may be attached to the linker by an activated ester.

In a further preferred embodiment, the set of oligonucleotide probes of the invention is characterized in that the label is attached to a non-nucleotidic unit.

One advantage of attaching labels to non-nucleotide units is that the design and synthesis of individual oligonucleotide probes is sequence independent. That is, all oligonucleotide probes can be designed so that they contain labels at the same position. Furthermore, contrary to what is generally accepted in the art, it was surprisingly found in the context of the present invention that the binding efficiency with the target sequence is still optimal when attaching labels to non-nucleotidic units.

As used herein, the term "non-nucleotidic unit" refers generally to any reagent or molecule which may conveniently permit the attachment of single or multiple moieties, e.g., labels or intercalators, to a nucleotide probe at any particular preselected position thereof. Non-nucleotide units according to the invention are preferably monomeric units which can be synthetically coupled with specific nucleotide monomeric units from a nucleotide reagent to produce a defined sequence polymer having a backbone comprising nucleotide monomeric units and non-nucleotide monomeric units. That is, the non-nucleotide units of the invention can be placed at any desired position within the stem sequence of nucleotides. The non-nucleotidic units of the invention may comprise a ligand which is a linker-arm portion with a label or which may participate in a conjugation reaction once the linker-arm is deprotected. The non-nucleotide units of the invention may further contain an azide or alkyne moiety suitable for click chemistry (Best, m.d., Biochemistry2009, 48 (28): 6571-. Suitable protecting groups that can be used to protect the linker arm functionality during formation of the polymer are known in the art.

The non-nucleotide unit according to the invention may further comprise two coupling groups to allow its stepwise inclusion into the polymer of nucleotide and non-nucleotide monomeric units, wherein one of such coupling groups is defined such that it can be effectively coupled to the end of a growing chain of monomeric units, the second coupling group being capable of further extending the growing chain of mixed nucleotide and non-nucleotide monomers in a stepwise manner. The non-nucleotidic units according to the invention are well known in the art and are described, for example, in EP 0313219.

In a further preferred embodiment, the oligonucleotide probe of the invention comprises a label suitable for detection by means of a chromogenic reaction, by means of a metallographic reaction, or by means of direct or indirect fluorescence analysis.

The term "chromogenic reaction" as used herein includes, but is not limited to, all standard methods known in the art that cause the formation of a chromophore at the site of enzyme activity. That is, in a simple single-step or multi-step chromogenic reaction, a colorless substrate can be enzymatically converted to a colored product. In the context of the present invention, a chromogenic reaction includes any reaction or collection of reactions that exhibit discrete regions, points or bands of enzymatic activity that can be studied, measured and/or analyzed by visual inspection. For example, detection of biotinylated oligonucleotide probes is typically by colorimetric or chemiluminescent visualization through avidin or streptavidin conjugated to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase (HRP). For example, 4-nitroblue tetrazoliumChloride (NBT) and 5-bromo-4-fluoro-3-indole-phosphate (BCIP) are well known in the art as efficient chromogenic substrates for alkaline phosphatase, which are converted to blue-colored products. For example, detection by chemiluminescence visualization may include any Enhanced Chemiluminescence (ECL) reaction involving light emission during horseradish peroxidase (HRP) -and hydrogen peroxide-catalyzed luminol oxidation. The emitted light may beCaptured on a membrane or by a CCD camera, can be used for qualitative or semi-quantitative analysis. Such detection systems are conventionally used in the art and are available, for example, from GE Healthcare (USA).

The term "metallographical reaction" as referred to herein generally refers to any type of enzymatic or non-enzymatic reaction in which metal is selectively deposited in the presence of a suitable metal source and activating reagent to give black, highly localized staining. That is, metallographical reactions refer to the deposition of enzyme-catalyzed or metal-catalyzed metals from solution. Preferably, the metallographical reaction of the enzyme according to the invention comprises the use of antibody-conjugated horseradish peroxidase (HRP) and silver (I) ions in solution. Alternatively, metal particles, such as gold particles, can be staged for highly specific silver deposition from a suitable silver salt solution in the presence of a suitable reducing agent. Preferably, the non-enzymatic metallographic reaction according to the invention comprises the use of oligonucleotide probes directly or indirectly labelled with gold particles and a silver (I) ion solution. Such metallographic reactions have proven to be highly sensitive to In Situ Hybridization (ISH) and Immunohistochemical (IHC) detection methods, where it readily visualizes endogenous copies of a single gene. Since it is used in a conventional bright field microscope, it does not require fluorescent optics or dark adaptation for the user. The signal is permanent and does not have the photobleaching problems associated with fluorescent dyes. Such detection systems (e.g., EnzMet^TM，NanoGold^TM) Are conventional in the art and are commercially available from a variety of suppliers including, for example, Nanoprobes Inc (USA).

The term "fluorescence assay" as used herein generally refers to all types of imaging methods known in the art that are suitable for visualizing, detecting, analyzing, and/or quantifying fluorescence signals. In particular, fluorescence analysis according to the present invention includes, but is not limited to, all known methods of overlapping and confocal fluorescence microscopy, including, for example, multiple fluorescence probe microscopy. The oligonucleotide probe may be directly labeled with a fluorescent dye and visualized with a fluorescence microscope (direct fluorescence analysis), or the oligonucleotide probe may be labeled with a hapten and detected with a fluorescently labeled antibody to the hapten, and then visualized with a fluorescence microscope (indirect fluorescence analysis). Indirect fluorescence analysis also includes the use of unconjugated antibodies in combination with fluorescently labeled secondary antibodies.

Thus, in a preferred embodiment, the set of oligonucleotide probes of the invention is characterized in that said label is a fluorescent label or hapten, in particular a fluorescein dye, a rhodamine dye or a cyanine dye, or a biotin, digoxigenin or a 2, 4-dinitrophenyl moiety.

Depending on the number of labels present in each individual oligonucleotide probe, a set of oligonucleotide probes comprises a variable total number of labels. For example, as exemplified in example 3, the best results are obtained when the set of oligonucleotide probes comprises a total number of labels of at least 1000 to 2000, preferably at least 4000 to 8000.

Thus, in a preferred embodiment, a set of oligonucleotide probes of the invention comprises a total number of labels of at least 1000, preferably at least 2000, more preferably at least 4000 and most preferably at least 8000.

By providing a set of oligonucleotide probes comprising a high number of labels in total, the number of labels incorporated in each individual oligonucleotide probe can be reduced, so that each label can be located within the molecule in an optimal manner. That is, by incorporating only a limited number of labels, for example, in the case of fluorescent labels, undesired quenching effects can be avoided, or the hapten can be placed so that subsequent detection is optimized for optimal antibody binding.

To date, oligonucleotide probes have been successfully used for the detection of high abundance target sequences, e.g., abundantly expressed mRNA or repetitive genomic sequences, including, e.g., centromere sequences, however the use of oligonucleotide probes for the detection of low abundance sequences has been described as disadvantageous (see, e.g., Jin et al, 2001; Morphology Methods: Cell and molecular biology Techniques: 27-46).

In the context of the present invention, however, oligonucleotide probes are successfully used to detect non-repetitive genomic sequences. Thus, oligonucleotide probes directed against non-repetitive regions are best suited for detecting genomic target sequences of interest.

Thus, in a preferred embodiment, a set of oligonucleotide probes according to the invention is complementary to a non-repetitive region of a genomic target sequence.

One of the advantages of using sets of oligonucleotide probes complementary to non-repetitive regions of the genomic target sequence is that low abundance genes can be detected and/or visualized with high sensitivity.

The term "non-repetitive region" as used herein generally refers to any sequence in the genome of interest that is not a repetitive DNA element. In particular, a non-repetitive region according to the present invention refers to any region outside of a repetitive sequence of a genome (e.g., the human genome), including, but not limited to, centromere, telomere, tandem repeats (e.g., satellite DNA, mini-satellite DNA, and microsatellite DNA), and interspersed repetitive elements, such as SINE (short interspersed nuclear element) and LINE (long interspersed nuclear element). The repetitive elements have been found in most eukaryotic genomes analyzed, most present in multiple copies, and do not encode proteins or RNA. Interspersed repetitive elements are usually present as individual copies, widely distributed throughout the genome.

In further preferred embodiments, the set of oligonucleotide probes may be complementary to the sense or antisense strand of the genomic target sequence.

By using oligonucleotide probes complementary to the sense or antisense strand of the genomic target sequence, optimal hybridization is ensured since the probes can no longer anneal.

The term "sense" as used herein means that the sequence of the oligonucleotide probe is identical to the target sequence, e.g., identical to the coding strand of the genomic sequence. In contrast, the term "antisense" refers to the sequence of the opposite strand. In the context of the present invention, it has been found that oligonucleotide probes complementary to the sense or antisense strand of a genomic target sequence can be used to detect a genomic target sequence of interest.

The human epidermal growth factor receptor (HER-2) oncogene is a member of the epidermal growth factor receptor or erb gene family, encoding a transmembrane tyrosine kinase receptor, which is increasingly becoming a major classifier of invasive breast cancer and a target for cancer therapy. The HER-2(C-erbB-2) gene is located on chromosome 17 q. HER-2 has been linked to the prognosis and response of treatment with anti-HER-2-humanized monoclonal antibody (trastuzumab) in patients with advanced metastatic breast cancer. HER-2 status was also tested for its ability to predict breast cancer response to other treatments, including hormone therapy, topoisomerase inhibitors, and anthracyclines. Both morphology-based and molecular-based techniques have been used to measure HER-2 status in clinical samples of breast cancer. To date, Immunohistochemical (IHC) staining has been the predominant method used. However, unlike most IHC assays, the assessment of HER-2 status is quantitative, not qualitative, as HER-2 is expressed in all mammary epithelial cells. Studies have shown that there is an excellent correlation between gene copy status and protein expression levels when standard IHC analysis is performed on carefully fixed, processed and embedded samples.

In the context of the present invention, it was surprisingly found that the set of oligonucleotide probes of the invention is particularly suitable for the detection of the human epidermal growth factor receptor 2 (human HER-2) locus. Human epidermal growth factor receptor 2(HER2) is highly expressed in approximately 30% of breast cancer patients, and there is a large body of evidence supporting a relationship between HER2 overexpression and poor overall survival. Thus, there is always a need for an improved method for detecting normal or abnormal human HER-2 gene expression. For example, the detection of the human epidermal growth factor receptor 2 locus according to the present invention is exemplified in examples 1 to 3.

It was further found in the context of the present invention that the detection of the metallographic phase of a genomic target sequence is particularly advantageous when the set of oligonucleotide probes is directed against clustered regions of the target sequence of interest. The term "clustered" as used herein refers to different oligonucleotide probes directed against separate target sequences that may be in close proximity to each other. Such close proximity may include, but is not limited to, a distance of about 50 to 500 nucleotides. Clustering of oligonucleotide probes further means that these regions are not evenly distributed over the genomic target sequence of interest, but that the oligonucleotide probes may be unevenly distributed, covering regions within close proximity (i.e., clusters) within the locus as well as regions located at greater distances. Clustering of oligonucleotide probes according to the invention is illustrated in FIG. 3 (see, e.g., pools 2 and 4).

Thus, in another preferred embodiment, the set of oligonucleotide probes of the invention is characterized in that said oligonucleotide probes are clustered within the genomic target sequence.

In the context of the present invention, it was surprisingly found that the analysis and/or visualization of genomic target sequences is particularly sensitive when clustered oligonucleotide probes and metallographic detection processes are used.

In another preferred embodiment, the set of oligonucleotide probes of the invention is characterized in that the oligonucleotide probes are directed against a genomic target sequence, wherein the genomic target sequence comprises the human epidermal growth factor receptor 2 (human HER-2) locus.

The term "human epidermal growth factor receptor 2 (human HER-2) locus" as referred to herein generally includes the entire genomic sequence and/or genomic region constituting the gene encoding human epidermal growth factor receptor 2(HER 2). These sequences may or may not be necessary for gene expression. The sequences that make up the human epidermal growth factor receptor 2 (human HER-2) locus are known to those skilled in the art and can be identified, for example, by database searches, including but not limited to the NCBI (national center for biotechnology information) GenBank database.

In the context of the present invention, it was surprisingly found that clustering of oligonucleotide probes is particularly advantageous when applying metallographic detection methods. That is, clustering of oligonucleotide probes allows for highly sensitive detection of gene sequences, since it is hypothesized that enzyme-mediated deposition of metals is accelerated by the close proximity of labels to each other.

In the context of the present invention, it has been demonstrated that a set of oligonucleotide probes comprising at least 100 different oligonucleotide probes can be successfully used for the detection of a genomic target sequence, in particular a genomic target sequence comprising the HER-2 locus. For example, the detection of genomic target sequences, in particular target sequences comprising the HER-2 locus, according to the invention is illustrated in FIGS. 1 to 4.

Thus, in another aspect, the invention provides the use of a set of oligonucleotide probes of the invention for detecting a genomic target sequence.

In the context of the present invention, the term "detecting" generally refers to visualizing, analyzing and/or quantifying the binding of an oligonucleotide probe to its target sequence. Detection according to the present invention includes, but is not limited to, visual inspection of the probed sample, including the use of standard microscopy techniques as well as any type of fluorescence analysis. In particular, the visual inspection of the sample is performed after hybridization of the oligonucleotide probe to its target sequence. For radiolabeling, detection refers to exposing the probed sample to X-ray film, including the generation of autoradiograms. Preferably, the detection according to the invention is carried out by means of a chromogenic reaction, a metallographic reaction or by means of direct or indirect fluorescence analysis.

In the context of the present invention, it was surprisingly found that a set of oligonucleotide probes comprising at least 100 different oligonucleotide probes allows for a detection method which provides various advantages over the prior art. That is, using the set of oligonucleotide probes according to the invention, the genomic target sequence of interest can be (i) probed in reduced numbers, (ii) after reduced hybridization time, and (ii) compared to methods known and available in the artAnd/or (iii) detected with reduced background. Known standard methods for detecting genomic target sequences employ, for example, Chromogenic In Situ Hybridization (CISH) and immunohistochemical (EHC) techniques (Ventana Inform)^TM，Ventana Medical Systems Inc.，USA；SpotLight^TM，Zymed Laboratories Inc.，CA，USA)。

Thus in a further aspect, the present invention provides a method of detecting a genomic target sequence of interest, said method comprising the steps of:

a) incubating the set of oligonucleotide probes and the sample according to the invention under conditions conducive to binding of the set of oligonucleotide probes to the genomic target sequence of interest; and

b) detecting binding of the oligonucleotide probe to the genomic target sequence.

All aspects and embodiments defined above also relate to a method of detecting a genomic target sequence of interest.

The term "under conditions conducive to binding" generally refers to conditions under which a labeled oligonucleotide probe of the invention can hybridize to its target sequence, i.e., conditions under which an oligonucleotide probe can bind to its target sequence. In particular, in the context of the present invention, conditions that facilitate binding refer to the formation of complementary base pairs between the oligonucleotide probe and the target sequence. Hybridization between the oligonucleotide probe and the target sequence, and thus formation of complementary base pairs, is defined by hydrogen bonding and hydrophobic interactions in equilibrium. That is, annealing and separation of two complementary strands depends on a variety of factors, including temperature, salt concentration, pH, the nature of the probe and target molecule, and the composition of the hybridization and wash solutions. The optimum temperature for hybridization is preferably below T_mThe value is in the range of 15-25 ℃, the Tm value defines the melting temperature (T) of the hybrid_m) I.e., the temperature at which 50% of the double-stranded nucleic acid strands separate. Calculating T_mVarious formulas of values are known to those skilled in the art. RNA-RNA hybrids are generally more stable than DNA-DNA or DNA-RNA hybridsThe temperature is determined to be 10-15 ℃, so that more stringent conditions are required for hybridization and washing. Conditions that facilitate binding according to the invention also include the use of hybridization buffers containing reagents to maximize duplex formation and inhibit non-specific binding of the probe to tissue or cells. The concentration of the probes must be optimized for each probe and for each tissue. Conditions conducive to binding also refer to incubating the probe and target sequence for a sufficient time to allow optimal hybridization, including removal of unbound probe by application of one or more washing steps.

In particular, in the context of the present invention, the conditions conducive to binding according to the present invention refer to hybridization conditions in which the oligonucleotide probe is incubated with the target sample in solution. That is, the conditions conducive to binding do not refer to any conditions under which the oligonucleotide probes are incubated with the immobilized target sample, for example, conditions in the context of a DNA array. For example, conditions that facilitate bonding according to the present invention are described in example 3.

Analysis of gene expression can be indicative of the presence of a disease, for assessing different stages of a disease, or for success or failure of a therapeutic treatment. For example, HER-2 gene overexpression has been described as associated with high grade and broad forms of ductal carcinoma, while HER-2 gene amplification has been associated with poor outcome of invasive lobular carcinoma. In addition, HER-2 gene amplification and protein overexpression have been consistently associated with high tumor grade, DNA aneuploidy, high cell proliferation rates, negative analysis of nuclear protein receptors for estrogen and progesterone, p53 mutation, topoisomerase IIa amplification, and alterations in various other molecular biomarkers of breast cancer invasiveness and metastasis (see, e.g., Ross et al, 2004). Status of HER-2 Gene expression further correlated with the use of the anti-HER-2-humanized monoclonal antibody trastuzumab (Genentech, CA, USA) treatment.

Thus, in a preferred embodiment, the method for detecting a genomic target sequence according to the invention is further characterized in that said detection is for diagnostic purposes, in particular for human diseases and/or for the effectiveness of a therapeutic treatment in a patient.

The reliability of sensitive detection methods for diagnostic purposes as well as for assessing medical treatments is of particular importance.

In another preferred embodiment, the invention or the use of the method of the invention is characterized in that the detection is performed by means of in situ hybridization.

In situ hybridization is a standard method and is therefore of particular importance.

As used in the context of the present invention, the term "in situ hybridization" refers generally to any type of nucleic acid hybridization assay in which a labeled oligonucleotide probe is used to locate a particular DNA or RNA sequence in a tissue (in situ), a section or section of a cell, or throughout an organism if the tissue is small enough (e.g., plant seeds or drosophila embryos). Thus, in situ hybridization according to the present invention refers to any method of locating and detecting a particular nucleotide sequence by hybridizing the complementary strand of a nucleotide probe to the sequence of interest in a morphologically preserved tissue section or cell preparation.

There are generally two types of labeled probes that can be used in situ hybridization methods, i.e., oligonucleotides that are labeled directly or indirectly. An oligonucleotide may be indirectly labeled by conjugating it to a molecule that can be detected by a second molecule or an antibody, e.g., biotin. Biotin-labeled oligonucleotides are often used in situ hybridization techniques, for example, and may be detected by binding a fluorochrome-conjugated avidin to a biotin-labeled and hybridized probe. Another type of indirectly labeled oligonucleotide may include those conjugated to digoxigenin or dinitrophenyl. For example, these markers can be detected by fluorochrome-conjugated anti-digoxigenin or anti-dinitrophenyl antibodies. Other oligonucleotides can be directly labeled by covalently attaching a fluorescent dye to a single nucleotide or non-nucleotide linking reagent at a defined location, which eliminates the need to use a second detection molecule. Labeling of oligonucleotide probes after DNA synthesis is often easier than incorporating labeled nucleotides during synthesis. In contrast, detection of directly labeled probes is less time consuming, as the use of a second detection molecule may be omitted. Thus, there are clear advantages and disadvantages to both directly and indirectly labeled probes.

Preferably, the oligonucleotide probe of the present invention is labeled with dinitrophenyl and detected by using an anti-dinitrophenyl antibody.

The oligonucleotide probes used for in situ hybridization may be DNA or RNA or a combination of both, and may or may not be fluorescently labeled. Thus, the in situ hybridization of the present invention includes both Fluorescent In Situ Hybridization (FISH) and all known methods of Chromogenic In Situ Hybridization (CISH) techniques, which may be used in medical diagnostics to assess chromosomal integrity, for example. In situ hybridization can also be performed by using hapten or radiolabeled oligonucleotide probes, for example, where detection is performed by using fluorophore-labeled antibodies, avidin or X-ray exposure. In situ hybridization methods are well known to those skilled in the art, for example, in Silverman and Kool, 2007, Advances in Clinical Chemistry, Vol.43: 79-115.

The in situ hybridization according to the present invention further includes all known procedures of multichannel or multicolor in situ hybridization, i.e., all procedures in which oligonucleotide probes with fluorescent labels of different colors are used, or in which oligonucleotide probes with different haptens and different enzyme/substrate systems are used. These multiple labels may be detected simultaneously or in a series of detection steps. Such methods are known to those skilled in the art and are described, for example, in Wiegant et al, 1993(cytogene Cell Genet 63: 73-76).

Such as exemplified in example 1 of the present invention, in reduced time, i.e., at hybridization times of significantly less than 4 hoursThen, the detection of the genome target sequence is successfully realized. Furthermore, in contrast to methods known in the art (e.g., Ventana Inform @)^TMDetection method) improved results were obtained even after hybridization times of 1 hour or 30 minutes.

Thus, in a preferred embodiment, the method of the invention is further characterized in that the detection of step b) is by means of a chromogenic reaction, by means of a metallographic reaction, or by means of direct or indirect fluorescence analysis; and/or the incubation of step a) is completed after at most 4 hours, 3 hours or 2 hours, in particular after at most 1 hour or 30 minutes.

As surprisingly found in the context of the present invention, detection of genomic target sequences can be achieved in shorter hybridization times compared to standard detection methods known in the art (see, e.g., fig. 2). For example, detection of genomic target sequences by means of indirect fluorescence analysis is illustrated in FIG. 4.

The term "complete" as referred to herein is not to be understood as meaning that the incubation of step a) is essentially complete. It means that an increase in incubation time does not result in a significant improvement or increase in signal. In contrast, the term "complete" refers to the completion of hybridization of the oligonucleotide probe to the target sequence of interest such that a detectable result can be obtained.

It is further observed in the context of the present invention, for example as illustrated in example 3, that the selection of different subsets of oligonucleotide probes, resulting in various oligonucleotide probe "cocktail" compositions, allows for the hybridization with methods known in the art (e.g., Ventana Inform^TM) Compared to improved detection.

In a further aspect, the present invention provides a method of generating a set of oligonucleotide probes directed against a genomic target sequence of interest, said method comprising the steps of:

a) designing a set of oligonucleotide probes complementary to at least 100 different regions of a genomic target sequence of interest, in particular wherein said regions comprise non-repetitive sequences; and

b) a set of oligonucleotide probes according to the invention was synthesized.

All aspects and embodiments defined above also relate to a method of generating a set of oligonucleotide probes directed against a genomic target sequence of interest.

One of the advantages of the present invention is that oligonucleotide probes can be produced in a highly reproducible manner. In addition, the design of oligonucleotide probes can be adapted to a variety of different gene sequences.

The term "design" as used herein refers to the identification of different target sequences that are complementary to oligonucleotide probes. As detailed above, the oligonucleotide probes of the invention can be designed such that they are complementary to either the sense or antisense strand of the target sequence. Preferably, the oligonucleotide probes are designed such that they are not complementary to any repetitive region of the human genome. Design further means that the oligonucleotide probes can form any type of complementary base pair with the target sequence of interest, including, but not limited to, canonical and atypical base pairs and base mismatches. That is, the oligonucleotide probes of the present invention may preferably, but need not necessarily, be designed to exhibit 100% complementarity to the target sequence. Oligonucleotide probes can also be designed to show less than 100% complementarity to the target sequence if deemed appropriate. However, the complementarity between the oligonucleotide probe and the target sequence of interest must be sufficient to provide binding to, and thus detection of, the target sequence of interest. As the number of cloned genes increases, oligonucleotide probes can be readily designed based on any published cDNA sequences and/or gene bank entries. Databases of genomic sequences from various organisms are known to the person skilled in the art and include, for example, all public databases from NCBI (national bioinformatics center, USA).

The term "synthesis" as used herein preferably means the generation of oligonucleotide probes by means of chemical synthesis, including but not limited to the use of automated DNA and/or RNA synthesizers and phosphoramidite chemistry. Automated DNA or RNA synthesizers are routinely used by those skilled in the art and are commercially available from various suppliers, such as applied biosystems (Darmstadt, Germany), Biolytics (Newark, Calif., USA) or BioAutomation (Piano, TX, USA). Preferably, the oligonucleotide probes of the invention are synthesized in one or more 96-well plates, thus providing rapid and efficient high-throughput synthesis. Chemical synthesis of oligonucleotide probes in a 96-well plate format not only allows for rapid synthesis of a variety of different oligonucleotides, but also allows for efficient and combinatorial selection of different subsets of oligonucleotides. As already detailed above, the method of detecting a genomic target sequence of interest according to the invention can be further optimized by the individual and combined selection of at least 100 different subsets of oligonucleotides in a large pool of oligonucleotides produced by multiple 96-well format syntheses, which subsets can then be used to construct a set of oligonucleotide probes according to the invention. For example, the combinatorial selection of different subsets of oligonucleotide probes according to the invention is illustrated in example 3. This optimization process can be further evaluated through a readout of experiments.

Optionally, the oligonucleotide probes may be further purified by any type of standard method available and known in the art. Such methods include, but are not limited to, chromatographic methods, e.g., High Performance Liquid Chromatography (HPLC). Optionally, purification of the oligonucleotide probe is performed via one or more attachment protecting groups such as, for example, DMT (dimethoxytrityl). DMT groups are widely used for nucleoside protection in oligonucleotide synthesis. In principle, each oligonucleotide probe can be purified separately and/or individually. Preferably, the oligonucleotide probes of the invention are purified as a group or as a pool of different subsets. For example, an aliquot of the MWP probe pool consisting of 96 crude oligonucleotides can be purified on an RP 18HPLC column (Hypersil, 8 × 240mm) using a 0.1M triethylammonium acetate pH 7/acetonitrile gradient. DMT on the peak can be collected, desalted by dialysis, evaporated and dissolved in 10mM Tris pH8.0. OD260nm can be determined by UV detection.

In a preferred embodiment, the method of the invention is further characterized in that the synthesis of step b) comprises the incorporation of at least one label

i) Building blocks modified by phosphoramidites, or

ii) by an oxidation step during the synthesis of the phosphoramidite based oligonucleotide.

As used herein, the term "phosphoramidite-modified building block" generally refers to any chemical structure comprising a phosphoramidite or phosphoramidite derivative that can be used to introduce one or more labels into an oligonucleotide molecule. In particular, phosphoramidite modified building blocks can be used to successfully and routinely prepare single-or multi-labeled oligonucleotides directly on an automated synthesizer by covalently attaching building blocks to growing synthetic oligonucleotides by the same procedure as standard synthesis. Preferably, the phosphoramidite modified building block of the present invention refers to a deoxynucleoside phosphoramidite, e.g., an N4-aminoalkyl-2' -deoxy-cytosine nucleoside, or a C-5 substituted pyrimidine. The deoxynucleoside phosphoramidites can carry an N-protected aminoalkyl group at the C-5 position or at the C-4 position of a pyrimidine, at the C-8 position of a deoxyadenosine or deoxyguanosine, or at the C-7 position of a 7-deaza purine. Alternatively, the deoxynucleoside phosphoramidite can carry an azide or alkyne moiety suitable for click chemistry-based incorporation of the label. In particular, the phosphoramidite modified building blocks of the present invention further comprise a non-nucleoside moiety. Optionally, the phosphoramidite modified building block may be labeled prior to its incorporation into the oligonucleotide. Labels that may be attached to the phosphoramidite building blocks of the present invention include, but are not limited to, fluorophores, biotin, and 2, 4-Dinitrophenyl (DNP). Examples of fluorescently labeled phosphoramidite modified building blocks include, but are not limited to, fluorescein-dT phosphoramidite, cx-FAM phosphoramidite, and Quasar 570-dT phosphoramidite. These compounds are known to the person skilled in the art and can be obtained, for example, from various suppliers, such as Invitrogen (Carlsbad, Calif., USA), Biosearch Technologies (Novato, Calif., USA), biogenet Laboratories (San Ramon, Calif., USA) or Glen Research (Sterling, Calif., USA).

In a particularly preferred embodiment, the phosphoramidite-modified building block is 2, 4-Dinitrophenyl (DNP) -phosphoramidite.

2, 4-Dinitrophenyl (DNP) -phosphoramidite is a phosphoramidite linked to 2, 4-Dinitrophenyl (DNP). 2, 4-Dinitrophenyl (DNP) is an inexpensive small marker that can be detected immunogenically by using monoclonal IgG anti-DNP antibodies. The 2, 4-dinitrophenyl group can be incorporated into phosphoramidite linked oligonucleotides during solid phase synthesis. Various structures of DNP phosphoramidites are described in the art (see, e.g., Grzybowski et al, 1993, Nucleic Acid Research, Vol.21, No. 8: 1705-1712). To obtain maximum sensitivity in antibody detection, the DNP moiety is preferably attached to the phosphoramidite building block via a linker with variable size. The synthesis and antibody-mediated detection of oligonucleotides containing multiple 2, 4-dinitrophenyl reporter groups is well known to those skilled in the art and is described, for example, in Grzybowski et al, 1993.

The labels of the present invention may be further incorporated into the oligonucleotide probes by an oxidation step during phosphoramidite-based oligonucleotide synthesis. The process of oxidation during phosphoramidite based oligonucleotide synthesis is a process known to the person skilled in the art, for example, described in WO 2007/059816.

In a further aspect, the invention provides a kit comprising a set of oligonucleotide probes according to the invention, in addition, comprising at least one further component selected from the group consisting of: deparaffinization reagents, pretreatment reagents, washing reagents, detection reagents and product sheets.

It will be apparent to those skilled in the art that the kits of the invention may comprise a variety of standard components, for example, buffers, antibodies and/or reagents to stop a particular reaction. The skilled person will be able to adjust the composition of the kit to the main intended use, e.g. depending on the detection system, the cell or tissue under examination, the target sequence, the marker, the sample under examination, etc. In particular, some ingredients are illustrated below.

The term "deparaffinization reagent" as used herein generally refers to any type of substance suitable for removing wax from a wax-embedded biological sample. The deparaffinization reagent according to the present invention may comprise one or more paraffin-solubilizing organic solvents, one or more polar organic solvents, one or more surfactants, and may further optionally comprise water.

The term "pretreatment reagent" as used herein generally refers to any type of reagent or substance that, when incubated with a sample prior to adding an oligonucleotide probe to a hybridization buffer, can be used to block non-specific binding sites in solution, or is suitable for improving binding of the oligonucleotide probe to a target sequence.

The term "wash reagent" generally refers to any type of liquid composition that can be used to remove unbound oligonucleotide probes after hybridization. For example, the washing reagents of the invention may contain mono-or divalent salts, e.g., NaCl, buffer salts, e.g., sodium citrate, and/or formamide in various concentrations. In this regard, higher formamide concentrations and lower NaCl concentrations are generally used for higher stringency. Washing agents according to the invention are known in the art and are described, for example, in WO 93/06245.

The term "detection reagent" as referred to herein generally refers to any substance or reagent that is necessary or relevant for the detection of a labeled oligonucleotide probe set after hybridization to a target sequence. The detection reagents of the invention may include, but are not limited to, fluorescent or non-fluorescent labeled antibodies, substrates for enzymatic reactions, e.g., chromogenic substrates for horseradish peroxidase or alkaline phosphatase, or substances such as avidin or streptavidin.

The figures and examples below are intended to illustrate various embodiments of the present invention. Thus, the particular modifications discussed are not to be considered limiting of the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is therefore to be understood that such equivalent embodiments are to be included herein.

Drawings

FIG. 1 shows the visualization of HER-2 genomic gene amplification in xenografts from Calu3, ZR-75-1 and MCF7 cells, studied by in situ hybridization using DNP-labeled oligonucleotide probes at various concentrations. (A) There was no gene amplification. Left side picture: using the Ventana Inform^TMDetection of HER-2 genomic gene sequence by detection system (Cat. No.780-001, Ventana Medical Systems Inc., USA); VentanaHER-2/neu probe: a concentration of 10. mu.g/ml; right side frame: detection of the HER-2 genomic gene sequence using 1440 different oligonucleotide probe sets at a concentration of 4. mu.g/ml. Each oligonucleotide probe contains 81 nucleotides and 6 labels, respectively. Total number of markers: 8640. (B) low gene amplification. Left side picture: ventana Inform^TMA detection system; the concentration of the VentanaHER-2/neu probe was 10. mu.g/ml; right side frame: see (a), right panel. (C) High gene amplification. Left side picture: ventana Inform^TMA detection system; the concentration of the VentanaHER-2/neu probe was 10. mu.g/ml; right side frame: e.g., (A), right panel.

FIG. 2 shows the visualization of the HER-2 genomic gene sequence in xenografts from ZR-75-1 cells studied by in situ hybridization with DNP-labeled oligonucleotide probes using enzymatic metallographic detection after various hybridization times. (A) Hybridization time: 32 minutes; left side picture: using the Ventana Inform^TMDetection of the HER-2 genomic gene sequence of the detection system (Cat. No. 780-001; Ventana Medical Systems Inc., USA); right side frame: detection of the HER-2 genomic gene sequence using 1440 different oligonucleotide probe sets, each oligonucleotide probe comprising 81 nucleotides and 6 markers. MarkingTotal number of substances: 8640. (B) hybridization time: 1 hour; left side picture: using VentanaInform^TMDetection of HER-2 gene in the detection system; right side frame: see (a), right panel. (C) Hybridization time: 2 hours; left side picture: using the Ventana Inform^TMDetection of HER-2 gene in the detection system; right side frame: see (a), right panel.

FIG. 3 shows the visualization of the HER-2 genomic gene sequence in xenografts from ZR-75-1 cells studied by in situ hybridization with different DNP-labeled oligonucleotide probe sets containing various total numbers of incorporated labels. In situ hybridization was performed as described in FIG. 2. (A) Schematic representation of different sets of oligonucleotide probe compositions for detection of the HER-2 gene. Different subsets of oligonucleotide probes were generated by combining various oligonucleotide probes from different 96-well plates. Each box represents a 96-well plate containing 96 different oligonucleotide probes. (B) Use of a Ventana Inform containing approximately 15000 tags^TMIn situ hybridization of the detection system (upper left panel) was compared to the results obtained with different sets of oligonucleotide probes according to the invention. The oligonucleotide probes of the invention are generated in 96-well plates (15 plates in total), and different sets of oligonucleotide probes (e.g., different pools) are generated by combining different plates. Library 1: a combination of plates 1 to 15; total number of markers: 8640; library 2: combinations of plates 1 to 4 and 12 to 15; total number of markers: 4608; library 4: combinations of plates 1, 3, 5, 7, 9, 11, 13 and 15; total number of markers: 4608.

figure 4 shows the visualization of HER2 gene amplification in xenografts from MCF7 and Calu cells studied by in situ hybridization with DNP-labeled oligonucleotide probes using indirect fluorescent detection. Probes hybridizing to the HER2 locus were visualized using a Cy5 filter (Cy5 filter) and counterstaining was visualized by a DAPI filter (DAPI). Covering: cy5 and DAPI filters. (A) There was no gene amplification. DyLight was used in xenografts derived from MCF7 cells^TM649 detection of HER2 genomic sequence of conjugated anti-rabbit IgG. (B) High gene amplification. In a process fromIn xenografts of Calu cells, DyLight was used^TM649 detection of HER2 genomic sequence of conjugated anti-rabbit IgG.

FIG. 5 shows a schematic representation of exemplary labeled oligonucleotide probes for in situ hybridization. (A) Primary sequence and design of five different 87mer DNA oligonucleotide probes directed to the human HER-2 locus, each containing 81 nucleotides and 6DNP phosphoramidite labels (1 at the 5' end, 5 internally incorporated). The position of each label is indicated by an "X", wherein X represents 2, 4-Dinitrophenyl (DNP). The DNPs are located at positions 2, 19, 36, 53, 70 and 87, at intervals of 16nts, as counted from the 3' end. (B) Chemical structure of phosphoramidite modified building blocks linked to 2, 4-dinitrophenyl (X). DMP represents dimethoxytrityl.

Examples

Probe synthesis

Synthesis of 15 × 96-well plate DNA probes using 6 DNP-labeled 87mer oligonucleotides was performed on a Biolytic dr. oligo 192MWP DNA synthesizer using standard phosphoramidite chemistry and Biolytic's synthesis cycles at a 50nmol scale. The synthesis was performed in DMT on mode (DMT on mode). 2000A CPG dT support (Glen Research)^TMCat No.20-2032) on microwell synthetic plates (Orochem)^TMfilter plate 96well, cat No. of 1100). All other standard reagents required for phosphoramidite-based oligonucleotide synthesis were purchased from Prooligo^TMOr Glen Research^TMAcetonitrile from Baker^TM. The quality of oligonucleotide synthesis is frequently checked by color detection of the released DMT cations. After completion of the synthesis, the oligonucleotides were cleaved and deprotected with ammonia (20 h, 40 ℃) and then evaporated in a speed vac concentrator and resuspended in 600. mu.l of 10mM Tris pH 8.0. Thereafter, the concentration was determined by OD260nm measurement. Yield: 7-16OD260 nm. This crude mass was used to construct different probe libraries.

In situ hybridization

Use of Ventana for deparaffinization, pretreatment, washing and stringent washing^TMReagents, in situ hybridization analysis was performed on the Ventana Discovery XT system (cat. No. 750-701). Sample and Probe composition or Ventana INFORM HER2DNAProbe according to the invention^TM(Ventana, cat. No. 780-4332). Unless otherwise indicated, the set of oligonucleotide probes according to the invention consists of 1440 oligonucleotide probes, each comprising 81 nucleotides and carrying 6 non-nucleotide DNP labels at the same position, 16 nucleotides apart from each other. The total number of labels in this probe was 8640.

The probes are detected by enzymatic metallographic detection, by chromogenic detection or by indirect fluorescent detection. The probe composition according to the invention was diluted in hybridization buffer (containing formamide, dextran sulfate, sodium chloride, sodium citrate in Tris buffer) to a final concentration of 4. mu.g/ml. VentanaINFORM HER2DNA Probe as a stock solution in hybridization buffer at a final concentration of 10. mu.g/ml^TM(Ventana, cat. No.780-4332) was supplied by the manufacturer. The samples were formalin fixed paraffin embedded HER23 with 1 xenograft control slides (Ventana, cat. No. 783-4332). Each slide was provided with 3 tissue sections of xenografts from Calu3 (high HER2 gene amplified), ZR-75-1 (intermediate HER2 gene amplified) and MCF7 (no HER2 gene amplified) cells, respectively.

Slides were deparaffinized using EZ Prep (Ventana, cat. No.950-102) at 65 ℃ for 20 minutes followed by 4 times 4 minutes at 75 ℃. Slides were pretreated with reaction buffer (Ventana, cat. No.950-300) at 90 ℃ for 3X 8 min, then digested with protease 3(Ventana, cat. No.760-2020) at 37 ℃ for 20 min. Unless otherwise stated, the probes were hybridized at 52 ℃ for 2 hours. Stringent washes SSC (Ventana)^TMCat.no.950-110) at 78 ℃ for 3 × 8 minutes. The slides were then incubated with rabbit anti-DNP antibody (Ventana)^TMCat No.780-4335) was incubated at 37 ℃ for 20 minutes. For the experiment shown in FIG. 2, the protocol described above was modified so that the hybridization times were 32 minutes (A), 1 hour (B) and 2 hours (C), respectively.

Enzyme metallographic detection

Enzymatic metallographic Detection of the probes was performed using an ultra View SISH Detection Kit (Ventana, cat. No.780-001) prepared at 37 ℃ with an ultra View SISH-HRP (Ventana ultra View SISH Detection Kit)^TMCat No.780-001) for 16 minutes. The slides were then incubated with ultra View SISH Silver chromophore A, ultra View SISH Silver chromophore B and ultra View SISH Silver chromophore C at 37 ℃ for 12 minutes (Ventana ultra View SISH Detection Kit)^TMCat No. 780-001). Slides were counterstained with hematoxylin II (Ventana, cat. No.790-2208) at 37 ℃ for 4 minutes followed by bluring reagent (Ventana, cat. No.760-2037) at 37 ℃ for 4 minutes. The slides were then washed 2 ×, dehydrated in a series of alcohol dilutions (70%, 96%, 100%), and finally washed twice in xylene, then with Permount (Fisher Scientific)^TMNo. sp15-100) at cat.

Color production detection

Color production detection of Probe Ventana BlueMap^TMReagents (Ventana, cat. No. 760-120). Slides were incubated with alkaline phosphatase conjugated UltraMap anti-rabbit IgG (ventanacat. No.760-4314) for 12 min at 37 ℃ followed by incubation with Activator, BlueMapl and BlueMap2(Ventana, cat. No.760-120) for 20 min at 50 ℃. The slides were then washed 2 x in mild wash solution, dehydrated in a series of alcohol dilutions (70%, 96%, 100%), and finally washed twice in xylene, and then fixed with Permount (Fisher Scientific, cat. No. sp15-100).

Fluorescence analysis

Indirect fluorescence detection of probes Using DyLight^TM649 conjugated anti-rabbit IgG was performed at 37 ℃ for 20 min. Slides were counterstained with DAPI (Ventana, cat. No.760-4196) for 8 min at room temperature. The slides were then washed 2 x in mild wash solution, dehydrated in a series of alcohol dilutions (70%, 96%, 100%), and finally washed twice in xylene, then with Permount (Fisher Scientific, cat. No. sp15-100)) And (4) fixing. Fluorescence microscopy was performed using a fluorescence microscope (Leica DM5500) using a halogen fluorescent lamp (Leica el6000) and a Cy5 fluorescence filter cube (excitation 620nm, dichroic mirror 660mn, inhibition 700nm) for visualization of HER2 gene sequence signals and DAPI fluorescence filter cube (excitation 360nm, dichroic mirror 400nm, inhibition 470nm) for visualization of counterstaining. Images were captured with a CCD camera (Leica DFC 350FX) using a 60 × objective lens.

Example 1

The experiment shown in FIG. 1 demonstrates that at a hybridization time of 2 hours, when hybridized with Ventana INFORM HER2DNA Probe^TM(Ventana Medical Systems Inc., USA) the oligonucleotide probe set according to the invention produced similar signal intensities at lower probe concentrations (4. mu.g/ml versus 10. mu.g/ml).

Example 2

Using the set of oligonucleotide probes according to the invention, at lower concentrations (4. mu.g/ml vs. 10. mu.g/ml) with Ventana INFORM HER2DNA Probe^TMIn contrast, a more intense staining signal was observed after 32 minutes or 1 hour of hybridization. Although the oligonucleotide probe set according to the invention produced an optimal staining after a hybridization time of only 32 minutes, an INFORM was used^TMOptimal staining of HER2DNA Probe was only achieved after 2 hours, suboptimal staining was achieved after 1 hour (see fig. 2).

Example 3

The experiments listed in figure 3 show that clustering of subsets of oligonucleotide probes at both ends of the target locus (leaving the middle part of the target locus unlabeled) gave more intense staining when metallographic examination was used compared to the average distribution of subsets of oligonucleotide probes over the entire target locus (see figure 3, library 2 vs. library 4). Note, however, that the total number of markers is equal in both libraries. This clearly illustrates the advantage of the set of oligonucleotide probes of the invention in that it provides the possibility of designing the optimal combination of subsets of oligonucleotides to obtain the best signal intensity while minimizing the number of subsets of oligonucleotide probes necessary.

Example 4

The experiment shown in FIG. 4 reveals that a set of oligonucleotide probes according to the invention can be visualized using indirect fluorescence detection.

Claims (17)

1. A set of oligonucleotide probes comprising at least 100 different single-stranded oligonucleotide probes to a genomic target sequence of interest, wherein each individual oligonucleotide comprises at least one label.

2. The set of oligonucleotide probes of claim 1, wherein said set comprises at least 200, 300 or 400 different oligonucleotide probes, in particular at least 500 different oligonucleotide probes, more in particular at least 1000 different oligonucleotide probes.

3. The set of oligonucleotide probes of claim 1 or 2,

i) wherein each oligonucleotide probe has a length of 20 to 200 nucleotides, preferably 40 to 175 nucleotides, more preferably 60 to 150 nucleotides, especially 80 to 120 nucleotides, and/or

ii) wherein each oligonucleotide probe comprises at least two or three labels, in particular at least five labels, more in particular at least 10 labels, most in particular at least 15 labels.

4. The set of oligonucleotide probes of any one of claim 1,

i) wherein the set of oligonucleotide probes comprises one or more subsets of oligonucleotide probes, wherein each subset consists of oligonucleotide probes having a label at the same position, having the same sequence, or

ii) wherein the oligonucleotide probes comprise labels at approximately equally spaced positions, in particular at least 5, 10, 15, or 20 nucleotides apart, or

iii) wherein the oligonucleotide probe comprises labels at predetermined positions, optionally at positions located at a predetermined distance from each other.

5. The set of oligonucleotide probes of any one of claims 1 to 4, wherein the label is

i) Attached directly or indirectly through a linker to the base, sugar, or phosphate moiety of a nucleotide, or

ii) is attached to a non-nucleotidic unit,

in particular wherein the label is suitable for detection by means of a chromogenic reaction, by means of a metallographic reaction or by means of direct or indirect fluorescence analysis.

6. The set of oligonucleotide probes according to any one of claims 1 to 5, wherein the label is a fluorescent label or hapten, in particular a fluorescein dye, a rhodamine dye or a cyanine dye, or a biotin, digoxigenin or a 2, 4-dinitrophenyl moiety.

7. The set of oligonucleotide probes according to any of the preceding claims, wherein said set of oligonucleotide probes comprises a total number of labels of at least 1000, preferably at least 2000, more preferably at least 4000 and most preferably at least 8000.

8. The set of oligonucleotide probes of any one of the preceding claims,

i) wherein the oligonucleotide probe is complementary to a non-repetitive region of the genomic target sequence, or

ii) wherein the set of oligonucleotide probes is complementary to the sense or antisense strand of the genomic target sequence.

9. The set of oligonucleotide probes of any one of the preceding claims,

i) wherein the oligonucleotide probes are clustered within the genomic target sequence, or

ii) wherein the genomic target sequence comprises a human epidermal growth factor receptor 2 (human HER-2) locus.

10. Use of a set of oligonucleotide probes according to any one of claims 1 to 9 for the detection of a genomic target sequence.

11. A method of detecting a genomic target sequence of interest, the method comprising the steps of:

a) incubating the set of oligonucleotide probes and the sample of any one of claims 1 to 9 under conditions conducive to binding of the set of oligonucleotide probes to the genomic target sequence of interest; and

b) detecting binding of the oligonucleotide probe to the genomic target sequence.

12. The method of claim 11, wherein the detection is for diagnostic purposes, in particular for human diseases, and/or for the effectiveness of a therapeutic treatment in a patient.

13. The use according to claim 10, or the method according to claim 11 or 12, wherein said detection is performed by means of in situ hybridization.

14. The method of any one of claims 11 to 13, wherein

(i) The detection of the step b) is carried out in a color reaction mode, a metallographic reaction mode or a direct or indirect fluorescence analysis mode; and/or

(ii) The incubation of step a) is completed after at most 4 hours, 3 hours or 2 hours, in particular after at most 1 hour or 30 minutes.

15. A method of generating a set of oligonucleotide probes directed against a genomic target sequence of interest, the method comprising the steps of:

a) designing a set of oligonucleotide probes complementary to at least 100 different regions of a genomic target sequence of interest, in particular wherein said regions comprise non-repetitive sequences; and

b) synthesizing a set of oligonucleotide probes according to any one of claims 1 to 9.

16. The method of claim 15, wherein the synthesis of step b) comprises incorporation of at least one label, which is

i) Building blocks modified by phosphoramidites, or

ii) by an oxidation step during the synthesis of the phosphoramidite based oligonucleotide.

17. A kit comprising a set of oligonucleotide probes according to any one of claims 1 to 10 and, in addition, at least one further ingredient selected from the group consisting of: deparaffinization reagents, pretreatment reagents, washing reagents, detection reagents and product sheets.