Method for detection and localization of genes in situ using branched-DNA hybridisation

TECHNICAL FIELD

These Invention relates generally to nucleic acid chemistry and biochemical Tests. In particular, the invention relates to highly sensitive A method for in situ detection of a nucleic acid analyte. The method uses branched DNA oligo (Branched DNA oligo) and hybridization methods to determine the position of a nucleic acid to detect in a biological sample and identified.

BACKGROUND TO THE STAND OF THE TECHNIQUE

the Method of Branched DNA (bDNA-) signal amplification was in a microwell format used extensively to specific nucleic acid sequences to detect and quantify. Urdes et al. Branched DNA (BDNA) Technology, in: nonradioactive Analysis of Biomolecules, 388-395, C. Kessler (Ed.), Springer-Verlag, New York (2000). Naturally quantitative and highly reproducible, bDNA can to detection any nucleic acid targets be applied for known that without the use of radioactive probes have a sequence is.

It have been a number of bDNA assays for the quantification of viral nucleic acids developed. These include: the RNA of the human immunodeficiency virus type 1 (HIV-1), Kern et al., J. Clin. Microbiol. 34, 3196-3203 (1996); the RNA of the simian immunodeficiency virus (SIV), Sodora et al., AIDS Res. Hum. Retroviruses 14, 171-181 (1998); the DNA of the hepatitis B virus (HBV), Hendricks et al., Am. J. Clin. Pathol. 104, 537-546 (1995); the RNA of the hepatitis C virus (HCV), Detmer et al., J. Clin. Microbio. 34, 901-907 (1996); RNA Hepatitis G virus (HGV), Brandshagen et al., Am. J. Gastroenterol. 94, 1000-1005 (1999); and the DNA of cytomegalovirus (CMV) Chernoff et al., J. Clin. Microbiol. 35, 2740-2744 (1997).

In front a short time the bDNA technology has been used to drive the expression cellular to detect mRNAs and to measure, among other things: cytokines, Breen et al., Cell. Immunol. 178, 91-98 (1997), and Shen et al., J. Immunol. Methods 215, 123-134 (1998); Progesterone and estrogen receptors, Nargessi et al., Breast Cancer Res. Treat. 50, 47-55 (1998), and Nargessi et al., Breast Cancer Res. Treat. 50, 57-62 (1998); Insulin, Wang et al., Proc. Natl. Acad. Sci. USA 94, 4360-4365 (1997); glucokinase, . Cabrera Valladares et al, Endocrinology 140, 3091-3096 (1999); c-fos, Shyamala et al., Anal. Biochem. 266, 140-147 (1999); as aP2, Burris et al., Mol. Endocrinol. 13, 410-417 (1999). All these bDNA assays were developed to nucleic acids in serum, plasma or to measure cell lysates. However, none of these studies indicate a bDNA assay for the detection of nucleic acids in morphologically intact Cells or tissues toward or discloses such.

in the would contrast an in situ hybridization (ISH) test necessarily the ability specific nucleic acid sequences to detect in morphologically intact cells or tissues. The ISH methods were since the introduction of the policy by Pardue and Gall ago twenty years improved. Pardue et al., Proc. Natl. Acad. Sci. USA 64, 600-604 (1969). The use of non-isotopic probes for example, has the inherent eliminates problems associated with radioactive ISH methods were, as long orbital periods, the risk of exposure to radioactivity and waste disposal. additionally this has the incorporation of different target and Signalamplifikationssysteme The (SH-sensitivity improved. in spite of these advances the ISH method still has a number of challenges overcome will. These challenges include developing a sensitive and specific target detection, ensuring a precise Co-localization of signal and target, obtaining target sequences and obtaining cellular and tissue morphology. additionally to have ISH methods to address a number of practical concerns, among including ease of use, reproducibility and possible quantification, possible Automation, versatility and timely completion.

The catalyzed reporter deposition tyramide signal amplification (CARD / TSA) is one ISH method that has been proposed to some of these to address challenges and concerns. Some studies have shown that the CARD / TSA ISH method can detect 1-2 copies of HPV-16 DNA in SiHa cells. Siadat-Pajouh et al., J. Histochem. Cytochem. 42, 1503-1512 (1994) and Adler et al., Histochem. Cell. Biol. 108, 321-324 (1997).

A Study showed that it is possible is to adapt bDNA technology to an ISH format for <?page 3?>detection of mRNA. Cao et al., Proceedings of the American Association for Cancer Research, 89th meeting, New Orleans, LA (1998), and Antao Et al., In Situ Hybridization Using the bDNA Technology, in: Techniques in Quantification and Localization of Gene Expression, BK Patterson (Eds.), 81-93, Boston, Birkhauser Press (1999). Antao et al. describe the use of bDNA method for the in situ hybridization to RNA in individual cells. The main steps in the bDNA assay are prehybridization, sequential hybridization with target probes, Preamplifier-, Amplifier- and label probe, and finally signal generation and detection are. The test described in this study, however, has relatively low sensitivity.

It Therefore, there remains a need in the art, highly sensitive method for the in situ detection of nucleic acid analytes in biological provide samples. The present invention fulfills this and other needs, which exist in the art.

DISCLOSURE OF THE INVENTION

Accordingly it is a principal object of the invention, the above-mentioned requirements to meet in the art, by the Providing a method for the in-situ detection of a nucleic acid analyte in a sample of biological material based on bDNA hybridization of, wherein the method increased to a Sensitivity results.

the The method of the invention can be used to determine the position, ie subcellular Position of a nucleic acid identify within a single cell, based on a bDNA hybridization method.

additional Objectives, advantages and novel features of the invention are partially disclosed in the following description and to the skilled person recognizable partly by examination of the following or go from the implementation the invention produced.

correspondingly the invention is therefore a process for the in situ detection of a nucleic acid analyte provided in a sample of biological material comprising the implementation bDNA hybridization comprising the steps of: <ul><li>(A) preparing a sample of biological Material by:</li><li>(I) immobilizing the biological material on a substrate;</li><li>(Ii) permeabilizing the substrate-bound biological material by contacting the substrate-bound biological material with a solution, Proteinase K at a concentration of 0.5 ug / ml to 50 ug / ml;</li><li>(Iii) treating the sample with RNase to remove any RNA from the remove sample; and</li><li>(Iv) heating the permeabilized biological material to a temperature and a period of time which are effective to prevent any double-stranded DNA to denature;</li><li>(B) contacting the biological material with a target oligonucleotide probe under hybridizing conditions, wherein at least a portion the target probe to at least a portion of the nucleic acid complementary is, so an analyte-target probe complex is formed when the nucleic acid analyte is present in the sample;</li><li>(C) washing the biological material with a detergent washing fluid comprising at a temperature in the range of 21 to 60 ° C; and</li><li>(D) detecting any analyte-target probe complexes on the substrate by</li><li>(I) contacting the washed substrate and analyte-target probe complex with a preamplifier oligonucleotide probe under hybridizing conditions, wherein a first portion of the preamplifier to another Portion of the target probe is complementary as the portion of the Target probe, the nucleic acid analyte to complementary is, thereby forming an analyte-target probe-preamplifier probe complex is formed, if the nucleic acid is present in the sample;</li><li>(Ii) contacting the product of step (d) (i) with a Amplifier oligonucleotide probe under hybridizing conditions, wherein a first portion of the amplifier to a second portion the preamplifier complementary , whereby an analyte-target probe-amplifier probe complex formed , when the nucleic acid analyte is present in the sample;</li><li>(Iii) contacting the product of step (d) (ii) with a Labeling probe, conjugated to an alkaline phosphatase Oligonucleotide probe comprises, under hybridizing conditions, wherein a portion of the label probe to a second portion of the Amplifier probe binds, thereby forming an analyte-target probe-preamplifier probe-amplifier probe-label probe complex is formed when the nucleic acid is present in the sample;</li><li>(Iv) labeling the analyte-target probe-preamplifier probe-amplifier probe-label probe complex &lt;?page 4?&gt;With a detectable label; and</li><li>(V) detecting the presence of the label on the substrate, wherein the nucleic acid DNA, an endogenous gene or a segment thereof.</li></ul>

It is preferred that the nucleic acid from the group consisting of HIV DNA, CMV DNA, HPV DNA, LAP and IL-2 selected group is.

In a second embodiment is a method for identifying the position of a nucleic acid analyte provided in a sample cell of biological material, and based on bDNA hybridization. This method of includes locating or identifying the position of a nucleic acid the same steps as above with respect to the in-situ detection of a nucleic acid analyte described. Where the label is detected, however even so there the ID Case on the position of the analyte-target probe complex in one of the biological sample cell, the position of the nucleic acid in to cell.

In Alternatively, is a method for the detection of a nucleic acid in a sample of biological material, wherein the Process comprises carrying out includes bDNA hybridization to the nucleic acid analyte to detect in situ, wherein said method has a sensitivity possesses sufficient to from about 1 to about 10 copies of the nucleic acid detecting in the biological material.

BRIEF DESCRIPTION OF THE DRAWINGS

<figref idrefs="S38">1</figref> is a schematic representation of a bDNA ISH method according to the present Invention.

The <figref idrefs="S39">2A</figref>. <figref idrefs="S39">2 B</figref>. <figref idrefs="S39">2C</figref>. <figref idrefs="S39">2D</figref>. <figref idrefs="S39">2E</figref>. <figref idrefs="S39">2F</figref>. <figref idrefs="S39">2G</figref> and <figref idrefs="S39">2H</figref> are microscopic (60 × -) Pictures of product obtained in Example 1 results.

The <figref idrefs="S40">3A</figref>. <figref idrefs="S40">3B</figref>. <figref idrefs="S40">3C</figref> and <figref idrefs="S40">3D</figref> are microscopic (60 × -) Pictures of product obtained in Example 2 results.

The <figref idrefs="S41">4A</figref>. <figref idrefs="S41">4B</figref>. <figref idrefs="S41">4C</figref> and <figref idrefs="S41">4D</figref> are microscopic (60 × -) Pictures of product obtained in Example 3 results.

The <figref idrefs="S42">5A</figref>. <figref idrefs="S42">5B</figref>. <figref idrefs="S42">5C</figref>. <figref idrefs="S42">5D</figref> and <figref idrefs="S42">5E</figref> are microscopic images of results obtained in Example 4.

The <figref idrefs="S43">6A</figref>. <figref idrefs="S43">6B</figref> and <figref idrefs="S43">6C</figref> are microscopic images of results obtained in Example 5 results.

The <figref idrefs="S44">7A</figref>. <figref idrefs="S44">7B</figref>. <figref idrefs="S44">7C</figref>. <figref idrefs="S44">7D</figref>. <figref idrefs="S44">7E</figref>. <figref idrefs="S44">7F</figref> and <figref idrefs="S44">7G</figref> are microscopic images of results obtained in Example 6 results.

MODES FOR CARRYING OUT THE INVENTION

I. DEFINITIONS AND OVERVIEW

Before The present invention will be described in detail, it should be noted that this invention is not limited to the specific probes, reagents, is test formats or the like is limited, since these vary can. It is also to be noted that the terminology used herein only for the purpose of describing particular embodiments only and not as limiting is to be understood.

It cited must be that contained, as in this description and in the annexed claims used, the singular forms "a / a", "an" and "the / the /" include plural forms, if not clearly apparent from the context otherwise. Therefore, for example, a reference to "an analyte-target probe complex" includes two or more such Complexes, a reference to a "washing" step includes two or more such Washing steps and the like.

In this description and in the claims which follow, the following terminology in accordance with the below Standing disclosed definitions used.

&lt;?page 5?&gt;

"Oligonucleotide" is a generic for polydeoxyribonucleotides (Containing 2'-deoxy-D-ribose or modified Forms thereof), for Polyribonucleotides (containing D-ribose or modified forms thereof), and for any other type of polynucleotide which is an N-glycoside of a Purine or pyrimidine base, or modified purine or Pyrimidine base is to understand. The oligonucleotides can be single-stranded or double-stranded, Typically, they are single-stranded. The present in the Invention oligonucleotides used also include normally from about 2 to about 100 monomer units, more typically from about 2 to about 80 monomer units and more preferably from about 2 to about 60 monomer units.

Of the Term "nucleic acid analyte" refers to a single- or double Nucleic acid molecule encoding a Target nucleotide sequence contains. The nucleic acid analyte can come from a variety of sources such as biological fluids or solids, food, located around materials etc. The term "nucleic acid analyte" is used interchangeably herein with the term "analyte" is used.

As used herein, the terms "target region" or "target nucleotide sequence" refers to a probe binding Region in the nucleic acid analyte is included. The target region must be 400 bases long at least. The term "target sequence" refers to a sequence with which a probe, for example, a target oligonucleotide probe, under the desired Conditions to form a stable hybrid.

As used herein, the terms "probe" and "oligonucleotide probe" refer to a structure consists of an oligonucleotide as defined above, the nucleic acid sequence a includes, which is complementary to a portion of a target nucleotide sequence, at least another probe or both. The oligonucleotide regions of the probes can consist of DNA and / or RNA and / or synthetic nucleotide analogs.

one will appreciate know that the binding sequences have not have perfect complementarity have to, to provide stable hybrids. constitute in certain situations, be stable hybrids, where less than about 10% of the bases mismatches , said loops of four or more nucleotides are ignored. Accordingly, the term "complementary" refers to an oligonucleotide, the a stable duplex with its "complement" forms under test conditions, in general, if the homology is approximately 90% or more.

As used herein, the terms "biological sample" or "biological are Material "interchangeably used and relate to a sample of tissue cells or liquid, which was isolated from an individual, including, but not limited on, for example, plasma, serum, spinal fluid, Semen, lymph fluid, the external secretions of the skin, secretions of the respiratory tract, secretions of the intestinal tract, secretions of the genitourinary tracts, tears, saliva, Milk, blood cells, tumors, organs, and also samples of in vitro Zellkulturkonstituenten (Including, but not limited to, conditioned medium, which arises from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components). It it is preferred that the biological sample in the form of a liquid having, for example, tissues or cells in a fluid, although solid Fabric can be used. Preferred uses of the present Method are in detecting and / or quantifying nucleic acids as follows: (a) viral nucleic acids, such as from hepatitis B virus ( "HBV"), hepatitis C virus ( "HCV"), hepatitis G virus ( "HGV"), human Immunodeficiency virus ( "HIV"), the human Papillomavirus ( "HPV") and the herpes virus family, including herpes zoster (chickenpox), herpes simplex virus types I &amp; II, cytomegalovirus ( "CMV") and Epstein-Barr virus; (b) bacterial nucleic acids, such as Chlamydia, Mycobacterium tuberculosis, etc .; and (c) numerous human sequences of interest, lysosomal inter alia acid phosphatase ( "LAP"), interleukin-2 (IL-2) and interferon-γ (INF).

Of the Term "hybridization conditions" is intended those conditions with respect to time, temperature and pH and the necessary amounts and concentrations of reactants and reagents described, sufficient to the annealing of at least a portion of a probe with its complementary to enable sequence. As is well known in the prior art, the hanging Time, temperature and pH conditions, the required are to achieve the hybridization of the size of the hybridizing oligonucleotide probe, the degree of complementarity between the oligonucleotide probe and the target and the presence of other Materials from the hybridization reaction mixture. The actual Conditions for each hybridization step are required, according to the state techniques well known or can without undue experimentation be determined.

Typical Hybridization conditions include the use of solutions, to a pH of the &lt;?page 6?&gt;about 7 buffered to about 8.5, and at temperatures of about 30 ° C to about 55 ° C, preferably from about 37 ° C to about 55 ° C, for a Period of about 1 second to about 1 day, preferably from about 15 minutes to about 16 hours, and more preferably from about 15 minutes to about 3 hours is performed.

"Hybridization conditions" also require a effective buffer. It can be any buffer that is compatible, that is chemically inert, is with respect to the probes and other components, but nevertheless hybridization between complementarity base pairs possible be used. A particularly preferred buffer, herein referred to as "Hybridization Solution A," comprises, 3 × SSC, 50% formamide, 10% dextran sulfate (MW 500,000), 0.2% Casein, 10 g / ml poly A, 100 ug / ml denatured salmon sperm DNA, wherein 1X SSC 0.15 M sodium chloride and 0.015 M sodium citrate. Another particularly preferred buffer, referred to herein as "Hybridization Solution B," comprises 5 × SSC, 0.1 to 0.3% sodium dodecylsulfate, 10% dextran sulfate, 1 mM ZnCl₂ and 10 mM MgCl₂. wherein 1 × SSC is as defined above.

"Optionally" means that the Circumstances described in connection may apply or not, so that the description includes instances where the event occurs and instances, where this is not the case. So comprises eg "where appropriate heating the permeabilized biological material "cases where the permeabilized biological material is heated, and cases where not heating the permeabilized biological Mateial is.

the The method of the present invention is an in situ bDNA hybridization assay. As will be appreciated by the prior art, provide bDNA-based The method provides an effective way to amplify a signal represents the otherwise would not be detected by other methods. Of the Professional is the for the implementation required by bDNA-based test methods and materials familiar.

Short said at a bDNA amplification includes a target oligonucleotide probe two locations: one for specifically hybridizing with a portion of the nucleic acid and one for specifically hybridizing with at least one other Probe. additional Oligonucleotide probes with unique sites that are designed to are to specifically hybridize to different probes may be used are so repeated stages of hybridization occur and a branched DNA structure. The final oligonucleotide probe which are linked in the branched structure by annealing shall bear at least one detectable label in itself. Therefore leads the original target molecule to a variety of signals, whereby the signal for simple Detection is amplified. additionally this can to the relevant (s) Literature, texts and other references for a description of conventional bDNA methods are referenced. See for example Collins et al., Nucleic Acid Res. 25 (15), 2979-2984 (1997).

The present invention provides highly sensitive method for Detection of a nucleic acid based on in situ bDNA hybridization ready. have previous in situ bDNA hybridization method relatively insensitive results provided. The present However, the invention provides an in situ bDNA method that highly is sensitive, for example, with a sensitivity sufficient, about 1 to about 10 copies of a nucleic acid analyte in biological to detect material.

II. THE DETECTION METHOD

In a first embodiment the invention provides a method for in situ detection of a nucleic acid in a sample of biological material, based on bDNA hybridization.

the The method comprises the steps of (a) preparing the sample of biological Material, (b) contacting the biological material with a Target oligonucleotide probe under hybridizing conditions, (c) washing the biological mate rials and (d) of detecting any analyte-target probe complex on the substrate.

The Sample of the biological material is obtained through conventional methods and includes, for example, the biopsy and the biological fluid extraction. Since the method for In-situ analysis is intended, however, tissues, cells can or organelles rather than a lysate are analyzed. Upon receipt and the handling of tissues, cells or organelles must during the all tests are done carefully to ensure that the material remains substantially intact.

The biological sample for use in the present process, ie the tissue or cells, to be analyzed may be prepared from any body part submitted, while the biological material to&lt;?page 7?&gt;taken that it contains the analyte of interest. Suitable tissue for use in the present process include, for example, without limitation, tissue from the adrenal glands, bladder, bone marrow, brain, Breast, heart, colon, esophagus, intestine, kidneys, the liver, the lungs, the lymph nodes, the nerves, the ovaries, the Pancreatic, prostate, (striated) skeletal muscle, the smooth muscle, spleen, stomach, testicular, tonsil, the trachea and the uterus. Furthermore, cells taken from the same tissues be, for suitable methods described herein. In addition, cells As described above, from cell-containing fluids such as plasma, Serum, spinal fluid etc., receive. Initially must be preserved, the biological material or "fixed". Although, according to the prior art Numerous methods for the fixation of biological samples known are, it is preferred that the biological sample with formaldehyde is fixed. The biological material can with a 4 -% - formaldehyde solution 30 are combined on ice minutes. Alternatively, can also other fixative, such as alcohol, can be used. Once fixed, the biological material must be immobilized on a substrate. Suitable substrates include those materials, the immobilization a a tissue, a cell or organelle allow, while the morphology of the sample is maintained. The substrate material must also genschaft to the egg heat resistant be fixed. Furthermore, the substrate must be inert with respect on the reagents used. Preferred substrates include glass, eg a glass slide, although elastic plastic material can be used.

the biological material on the substrate may be conventionally using Procedures are immobilized. For cells, it is preferred that immobilizing the biological material by using a centrifuge is. In general, the for immobilizing the biological material on the substrate required Force at about 200 × g to about 500 × g (Times gravity). It is preferred that the force used at about 300 x g lies.

The Immobilization of tissue samples is preferably by cutting very thin Sections of the tissue sample and placing these on a suitable Substrate performed. Preferably, the tissue is first placed in a cryostat and cooled to about -15 ° C to about -20 ° C to to freeze the tissue. subsequently is a microtome used in the frozen tissue sections to cut. Thereafter, a single section on a substrate placed, for example a glass slide, and it is the portion allows on the substrate to "melt", whereby the sample is immobilized. Instead frozen tissue tissues may be used, which were with wax, for example paraffin, infused. How well appreciate White, can Other methods for immobilizing the tissue sample may be used.

Around ensure that the oligonucleotide probes have access to the internal have around, it is necessary, the substrate-bound biological to make material permeable. It has been found that the Contacting the substrate-bound biological material with a Solution, containing Proteinase K at a concentration of about 0.5 ug / ml to about 50 ug / ml, preferably from about 5 ug / ml to about 20 ug / ml (A stock solution with an activity of 600 units per ml), the biological material to a sufficient extent permeable making so that the probe can pass through biological membranes freely, while the Morphology of the sample is maintained. The time and temperature parameters the permeable-making of the biological material are well known or can be determined experimentally. A reaction time of about 10 minutes at about 37 ° C is preferred for permeabilizing with Proteinase K.

If the nucleic acid double Comprises DNA, it is necessary to denature the DNA, so that probe hybridization by exposing the sample to a Heat treatment can take place, with a temperature and for a Period of time that is effective to prevent any double-stranded DNA denature. It has been found, however, that the heating of the DNA from about 1.5 minutes to about 5 minutes is sufficient to double-stranded DNA cellular under conservation denature components. The temperature may be any its temperature, which is known in the prior art, and sufficient to denature DNA. However, it is preferred that the temperature at from about 70 ° C to about 92 ° C, preferably at about 80 ° C to about 92 ° C, is located, a temperature of about 92 ° C is particularly preferred.

Additionally it is also necessary, when the nucleic acid analyte comprises DNA, any RNA which may be present, to digest. By digestion of the RNA can the probes do not hybridize to the RNA, whereby the potential false positives are minimized. In general, any any method which according to the prior art for the digestion is known from RNA, may be used. It is preferred, however, that RNase (available is used in Sigma, Fluka, etc.). RNase may be added to the sample in&lt;?page 8?&gt;one Amount of about 25 ug to about 100 ug (Per spot on slide) added be, and it may allow you be, for a certain period of time, and incubating at a temperature sufficient to digest the RNA. It is preferred, however, that about 40 ug (Per spot on slide) RNase added to to the sample and be maintained at one hour 37 ° C.

Other A process for the preparation of the biological material are available. For example, using commercially available kits, such as the ThinPrep^®-Objektträgersystem (Cytyc Corporation, Boxborough, MA).

once prepared, the biological material under hybridizing conditions, brought into contact with a target oligonucleotide probe. The target probe has a portion which is complementary to at least a portion the target sequence of the nucleic acid is. The nucleic acid present of interest in the sample, hybridize the nucleic acid and the target probe to form an analyte-target probe complex.

The Amount of added Target probes can be determined experimentally, but it is preferred that about 0.1 pmol be added pmol to about 10 (Per spot on slide) and allowing incubating at 40 ° C for about 3 hours. A preferred reaction medium for this hybridization step is the hybridization solution A (defined above).

is once passed a sufficient incubation period, so be, if present, both the substrate and the analyte-target probe complex washed to facilitate the removal of unbound target probes. The washing step requires the use of a washing liquid, which is generally a detergent and generally comprises a buffer solution.

The buffer solution Any conventional solution be known according to the prior art and for the removal non-hybridized oligonucleotide probes is suitable. Preferred buffer solutions include alkali metal salts. Particularly preferred buffer solutions Sodium chloride, sodium citrate and combinations thereof.

the Detergent is preferably a non-ionic detergent. Additionally is preferred when the detergent is also a hydrophilic surfactant. examples for Detergents include polyoxyethylene-based detergents, for example, BRIJ^® and TRITON^®,

Similar Detergents for the use is useful in the present invention are under the trade name TWEEN^®, GENAPOL^®, IGEPAL CA^®. Thesit^® and LUPROL^® sold (All available from commercial suppliers available).

is Once the washing liquid determined, the washing step is at least one, preferably two Times and particularly preferably three times performed. It it has been found that the temperature of the wash step, the affects sensitivity of the test. Therefore, work for the present Process temperatures in the range of about 21 ° C to about 60 ° C and good are used. Optimally, the wash step at room temperature performed.

is finished the washing step once, so missing unhybridized Target probes. Analyte-target probe complexes on the substrate are then detected to determine the presence of the nucleic acid to determine. Accordingly, additional oligonucleotide probes added, so that a branched network is formed: Has once formed a branched network, a plurality of detectable Marks added, namely with the effect of "amplifying" the signal for easier Detection. Therefore, the detection of an analyte-target probe complex by following reaches: <ul><li>(D) (i) contacting of the washed substrate and analyte-target probe complex with a preamplifier oligonucleotide probe under hybridizing conditions, wherein a first portion of the preamplifier to another Portion of the target probe is complementary as the portion of the Target probe, the nucleic acid analyte to complementary , whereby an analyte-target probe-complex formed , when the nucleic acid analyte is present in the sample;</li><li>(D) (ii) contacting the product of step (d) (i) with a Amplifier oligonucleotide probe under hybridizing conditions, wherein a first portion of the amplifier to a second portion the preamplifier complementary is, thereby forming an analyte-target probe-preamplifier probe-amplifier probe complex is formed when the nucleic acid is present in the sample;</li>&lt;?page 9?&gt;<li>(D) (iii) contacting the product of step (d) (ii) with a label probe, conjugated to alkaline phosphatase a Oligonucleotide probe comprises, under hybridizing conditions, wherein a portion of the label probe to a second portion of the Amplifier probe binds, thereby forming an analyte-target probe-preamplifier probe-amplifier probe-label probe complex is formed when the nucleic acid is present in the sample;</li><li>(D) (iv) labeling the analyte-target probe-amplifier probe-label probe complex with a detectable label; and</li><li>(D) (v) detecting the presence of the label on the substrate.</li></ul>

Each the probe hybridization steps, ie, steps (d) (i), (d) (ii) and (d) (iii) is, as described above, under conventional Hybridization conditions performed. For these particular hybridization steps However, it is preferred that each step at about 55 ° C with a Incubation period of approximately 25 minutes occurs. It is also preferred, that to each probe, ie, preamplifier, amplifier probe and Label probe hybridization solution B separately (defined above) is added, and indeed for contacting that probe with the growing complex. Therefore, as the amplifier probe is admixed with the hybridization solution B and placed in contact with the analyte-target probe-preamplifier probe complex, to form an analyte-target probe-preamplifier probe-amplifier probe complex.

Although the amount of the admixed probes using procedures which are known in the prior art, can be determined, it is preferred that the preamplifier and the amplifier probe each in an amount of about 1 fmole to about 10. pmoles (per spot on the slide) added will. these probes are particularly preferred in each case in a Amount of about 1 fmol to about 100 fmoles (per spot on slide) added.

The Marking is achieved when the label probe to the analyte-target probe-preamplifier probe-amplifier probe complex hybridized. The label probe includes one or more detectable labels, directly or indirectly provide a detectable signal. The labels may be bound covalently or non-covalently, specifically to the label probe as individual parts of the complementary sequence or may be as terminal part or terminal tail having a plurality of Marks occur. Various means have been used to provide of labels that are attached to a probe in the literature described. See, for example, Leary et al., Proc. Natl. Acad. Sci. USA 80, 4045 (1983); Renz et al., Nucl. Acids Res 12, 3435 (1984). Richardson et al., Nucl. Acids Res 11, 6167 (1983). Smith et al., Nucl. Acids Res 13, 2399 (1985). Meinkoth et al., Anal. Biochem. 138, 267 (1984).

markings which can be used, include Fluoreszenzagenzien, Chemilumineszenzagenzien, dyes, Enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and the same. Illustrative specific labels inter alia, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, α-β-galactosidase, Horseradish peroxidase and alkaline phosphatase. It is at least an alkaline-phosphatase label used.

The Detection of the detectable label can be by any according to the prior art known method may be reached and depends on the way from the mark. For Fluoreszenzagenzien is a large number of fluorometers available. For Chemilumineszenzagenzien are luminometer or films available. With enzymes, a fluorescent, chemiluminescent or colored Product will be provided, and it can fluorometrically, luminometer, spectrophotometrically or visually (preferably with the aid of a Microscope) can be determined. For the present method, it is preferred that the alkaline phosphatase substrate is added, to the presence of the alkaline phosphatase label using a bright-field or fluorescence microscopy to detect.

in the Unlike the previous tests, the present method for detecting a nucleic acid analyte highly sensitive. Previous tests based on the presence of multiple - often hundreds - copies the nucleic acid analyte. The present method is designed to relatively few copies to detect. Therefore, it is preferred that the method provides a sufficient having sensitivity to from 1 to about 10 copies of the nucleic acid to detect. However, it is particularly preferred that the The method has a sensitivity sufficient to provide from 1 to about 2 copies of the nucleic acid to detect.

III. SYNTHESIS OF THE PROBES

The Sequences of the probes are determined using standard procedures determined by the &lt;?page 10?&gt;are known to the prior art. Therefore, for example, the target probe sequence using a known sequence the analyte of interest is determined, specific for that analyte is. Those regions of the sequences involved in binding to be (and therefore to a different sequence of oligonucleotides complementary are - either a probe or analyte), comprise at least 15 nucleotides, usually at least 20 nucleotides and no longer than about 100 nucleotides. Typically, the binding sequences are approximately 25 nucleotides in length. They will normally be chosen to be on different Sequences of the analyte and / or to specific and different portions bind the various probes. In addition, have the oligonucleotides for a any number of probes optimally the same melting temperature.

probes with a second binding sequence are selected to be substantially complementary to the to be appropriate portion of the probe. The second binding sequence may be adjacent to the first binding sequence or by from this a non-complementary Cutscene be separated. The probes can be other non-complementary sequences contain, if desired. These non-complementary sequences allowed However, do not hinder the binding of the binding sequences or nonspecific binding result.

The probes can be prepared by oligonucleotide synthesis or by cloning, the former being preferred. Referring now to the beach art is well known to include processes for the synthesis of oligonucleotides Typically, the sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5 'hydroxyl group a growing oligonucleotide, wherein each addition by the nucleophilic attack of the terminal 5'-hydroxyl of the growing chain to the 3 'position the added running monomer is, which is typically a phosphorus derivative such as a phosphotriester, Phosphoramidite, or the like, is.

IV. ISOLATION

The present invention also provides a method for determining the Location or position of a nucleic acid analyte in a cell ready. indicated as above, this method comprises the same Steps as in situ detection, adds, however, a further step (e) of identifying the position of the Analyte-target probe complex in the biological sample cell as an indicator of the position of the nucleic acid in the cell added. Previous in situ detection tests were less accurate when Demonstrating the subcellular Position of the signal. The signal already carried out in situ tests could eg not in subcellular have been "contained" compartment. Alternatively, For this purpose, the signal of previous in situ assays over a relatively large have been spread portion of the cell, creating a definitive Conclusion with respect to the localization of the nucleic acid impossible is made. As temperatures for hybridization low are, ie, generally 55 ° C not exceed, and the sample is not subjected to rough treatment, makes the present method the ability possible, to determine the position of a single analyte within a cell. Even then, if the temperature for certain steps is increased, for example to about 92 ° C, namely in the denaturation of the DNA by heating, retains the present Method still the ability wherein, to determine the position of a single analyte within a cell.

V. BENEFITS

the This method can we use rden to any DNA, endogenous to detect genes or a segment thereof, known for having the sequence is. Preferred analytes for the method is particularly well suited include, but are not limited to, HIV DNA, CMV DNA, HPV DNA, LAP, IL-2 and gene transcripts.

in view of its sensitivity, ease of use, versatility, the reliability and the rapidity (results are obtained within a day) the present method has wide application areas in the early Detection of cancers and infectious diseases. The single-copy detection is particularly suitable for viral diseases such as HIV and numerous other advantageous since the diagnosis much earlier can be provided (if the viral load is still relatively low) which earlier Intervention and treatment possible is. additionally to the present method has applications in the field of Therapy selection. The present process can for example be adapted, to quickly and accurately the number of gene copies of the human epidermal Growth factor receptor 2 (/ neu HER2) to detect. This is for the Decision of benefit if started with an anti-HER2 therapy should be, for example, monoclonal anti-HER2 antibody therapy (Herceptin^®. available Genentech, Inc., South San Francisco, CA). Furthermore, the present method by modifying cellular or tissue pretreatment conditions ent&lt;?page 11?&gt;neither mRNA or DNA detected with the same number of probes.

the present method is highly specific and allows for exact localization of the target sequence in the cell when a portion of known of the nucleic acid analyte is. Furthermore, a localization is possible even if there is only one Copy of the nucleic acid are in the cell. Therefore, such a test has a wide application framework and is particularly useful in research and medicine useful. In the context gene therapies example, it is now possible to determine whether the Drug penetrates into the cytoplasm and / or nucleus.

In addition to the detection of cancers and infectious diseases and the Assisitieren in the therapy of choice has the capability of nucleic acids in detect tissue sections using the present methods, Other advantages. The shows in situ detection in a tissue sample for example, the extent to a cancer or an infectious pathogen surrounding the to the Tissue regions has spread. This information serves as a prognostic Indicator and is the basis for the capability to provide a suitable therapeutic approach tailor the disease.

Of the Scope of the invention defined by the claims contained in the Annex.

EXPERIMENTAL PART

The following examples are presented to the person skilled a perfect Disclosure and description about to send as the transact disclosed and claimed are to establish connections and use. There were efforts made to ensure accuracy with respect to numbers (eg Amounts, temperature, etc.) ensure nevertheless should some Errors and deviations are involved. If not, however, otherwise indicated, temperature is in ° C, and the pressure is at or near atmospheric pressure at the height sea ​​levels.

If not otherwise indicated all starting materials and reagents were obtained commercially (for example from Aldrich, Sigma and ICN) and without further Purification. There were Standardzellkultur- and cell harvesting methods used.

EXAMPLE 1

DETECTION OF HPV DNA BY BDNA ISH IN CELLS

A. Materials and Methods

i. Cell Culture

There they different strains included and amounts of human papilloma virus (HPV), which were human cervical carcinoma cell lines HeLa, CaSki and SiHa used to evaluate the present invention. Table 1 describes each cell line. Table 1<tables><table frame="all"><tgroup cols="3" colsep="1" rowsep="1"><colspec colname="1" colwidth="1*" /><colspec colname="2" colwidth="1*" /><colspec colname="3" colwidth="1*" /><tbody><row><entry colname="1">Cell line *</entry><entry colname="2">viral strain</entry><entry colname="3"> DNA copies per cell</entry></row><row><entry colname="1">CaSki</entry><entry colname="2">integrated HPV 16 DNA</entry><entry colname="3">400-600 copies</entry></row><row><entry colname="1">HeLa</entry><entry colname="2">integrated HPV 18 DNA</entry><entry colname="3">10-50 copies</entry></row><row><entry colname="1">SiHa</entry><entry colname="2">integrated HPV 16 DNA</entry><entry colname="3">1-2 copies</entry></row><row><entry colname="1">C33A (Control)</entry><entry colname="2">HPV-negative</entry><entry colname="3">0 copies</entry></row></tbody></tgroup></table></tables><ul><li>* All cell lines were obtained from American Type Cell Culture Collection (ATCC, Manassas, VA) and maintained in bottles cultured under conditions which is generally available from the ATCC are proposed. If necessary were the cells from the flasks using a mild trypsin treatment replaced.</li></ul>

ii. oligonucleotide probes

The HPV-16-specific target probes consisted of a total amount 26 DNA Oligonucle&lt;?page 12?&gt;otidsonden that about 90% of the E6 and E7 regions the HPV genome cover. These probes were first by creating a degenerate Consensus sequence created on 23 sequences for the E6 and E7 genes of the HPV-16 genome based, of GenBank using the Genetics Data Environment software (Harvard Genome Laboratory, Cambridge, MA) was obtained.

Potential Target probe sets were then using the sample Designer^®-Software (Bayer Diagnostics, Emeryville, CA) is produced, whereby a flexible Design of bDNA probe rows with minimal contribution to background allows and is thereby determined a constant melting point of 63 ± 2 ° C is. There were Endsonden after screening the probe rows possible Interaction with the other 39 HPV genotypes using a HybSimulators^® selected (Advanced Gene Computing Technologies, Inc., Irvine, CA) and human genomic DNA sequences using Blast2^TM (National Institutes of Health, Bethesda, MD).

HPV-18-specific Target probes consisted of a total amount of 32 DNA oligonucleotide probes, 90% of the E6 and E7 regions of the HPV genome covering. These Probes were prepared as described above, using a degenerate consensus sequence based on four sequences for the E6 and E7 genes of the HPV-18 genome and a constant melting temperature of 63 ± 2 ° C done.

Other DNA oligonucleotide probes, including the Preamplifier-, Amplifier- and alkaline phosphatase (AP) conjugated label probes, were extensively described. Collins et al., Nucleic Acids Res. 25, 2979-2984 (1997). In order to reduce the non-specific hybridization, were the unnatural Nucleotide 5'-methyl-2'-Desoxyisocytidin (IsoC) and 2'-Desoxyisoguanosin (IsoG) in the binding sites of the target, Preamplifier-, Amplifier- and AP-conjugated label probes included.

B. DNA detection

For the DNA detection The harvested cells were fixed with 4% formaldehyde in a phosphate buffer solution (PBS; 0.01 M phosphate buffer, pH 7.5), and that for 30 minutes to Ice, washed twice for 2 minutes each and resuspended in PBS. 200-ul aliquots the fixed cell suspensions were tospintrichter in doppeltgefleckte Cy pipetted (Shandon; Pittsburgh, PA) and for 6 minutes at 1500 U / min in a cytospin centrifuge (Shandon) using a calculated force equivalent 300 xg centrifuged. The cells were grown on slides through a graded Range of 70%, 90% and 100% ethanol at room temperature (RT) per dehydrated for 2 minutes at RT for 10 minutes air-dried and at -80 ° C for up to 2 stored weeks. The cells were grown on slides through a graded Series of 100%, 90% and 70% ethanol at RT for 2 minutes each again saturated with water and twice for 2 minutes each in PBS. The slides were at 37 ° C for 1 hour at 40 ug / ml RNase (Sigma) in 2 x SSC incubated (1 × SSC is 0.15 M NaCl, 0.015 M Na citrate) twice for 2 minutes each washed in PBS and in a preheated glass containing 7.5 to 10 ug / ml Proteinase K (Boehringer Mannheim, Indianapolis, IN) in PBS at 37 ° C for 10 minutes immersed. After two, 2 minutes each wash in PBS, cells were on slides through a graded Range of 70%, 90% and 100% ethanol at RT for 2 minutes each dehydrated and at RT for 10 minutes to air dry.

After the pretreatment, the cells were incubated with a prehybridization solution with an array of oligonucleotide target probes (above-described) hybridized, followed by hybridization with a series of oligonucleotide probes for the signal amplification. As a prehybridization everyone was Spot on the slide with 150 ul hybridization solution A incubated (3 × SSC, 50% formamide, 10% dextran sulfate [MW 500.0000], 0.2% casein, 10 ug / ml poly A, 100 ug / ml denatured salmon sperm DNA), namely at RT for 30 minutes. Slides were in a humidity chamber (Hybaid Omnislide instrument, Phenix Research Products, Hayward, CA) transferred, at 92 ° C for 5 minutes incubated and then removed from the chamber and at RT for 5 minutes on the bench cooled. The prehybridization solution was removed and each spot on the slide was incubated with 100 ul of hybridization solution A, containing 0.6 pmole HPV-specific target probes, namely at 40 ° C for 3 hours in the humidity chamber. After incubation with target probes the slides were at RT with a decreasing series of SSC buffers containing 0.0025% BRIJ^®- 35-detergent (SURFACT-AMPS^® 35, 10% Pierce, Rockford, IL) containing, washed as follows: 3 times 1-2 minutes with 2 × SSC; 3 times for 1-2 minutes with 0.2 × SSC; once for 5 minutes with 0.1 × SSC; and once for 2 minutes with 2 × SSC. Each spot on the slide was with 100 ul hybridization solution B incubated (5 x SSC, 0.1 to 0.3% sodium dodecyl sulfate [SDS], 10% dextran sulfate, 1 mM ZnCl₂, 10 mM MgCl₂) containing 90 fmoles preamplifier, namely at 55 ° C 25 minutes in the humidity chamber. The slides were washed twice in 0.1 × SSC, 1 mM EDTA for 1 minute and 4 minutes and then washed in 100 ul of hybridization solution B incubated, ent&lt;?page 13?&gt;holding 90 fmol Preamplifier, namely at 55 ° C 25 minutes in the humidity chamber. The slides were washed twice in 0.1 × SSC, 1 mM EDTA for 1 minute and 4 minutes and then washed in 100 ul of hybridization solution B incubated, containing 90 fmoles AP-conjugated label probe, namely in 55 ° C 15 Minutes in the humidity chamber. After washing in wash solution D (100 mM Tris (hydroxymethyl) aminomethane, pH 8.0, 0.1% BRIJ^® 35, 1 mM ZnCl₂, 10 mM MgCl₂) at RT for 5 minutes, 50 .mu.l buffered AP substrate (Fast Red, no. K597, DAKO Corporation, Carpinteria, CA) was added and the slides were for 10 minutes according to the manufacturer's instructions at RT incubated. Slides were three times rinsed each 3 minutes with water and then 40 Seconds with either Gills 1 hematoxylin (American Histology Reagent Company, Inc., Modesto, CA) or 0.0001% Bisbenzimid (Nr. B2883, Sigma) counterstained. Slides were ULTRAMOUNT^® (DAKO Corporation), Permount^® (Fisher offset Scientific) or 75% glycerol and stored at RT. The slides were measured using a Nikon E800 fluorescence microscope with a FITC or triple-band pass filter (Nikon, Foster City, CA) or with a 60 × objective -Hellfeld looked at, and the images were obtained using a cooled Optronics 3-chip color CCD camera (Optronics Engineering, Goleta, CA) recorded. The fluorescent or chromogenic images were analyzed using Image-trial software (Media Cybernetics, Silver Springs, MA) recorded. <figref idrefs="S38">1</figref> is a schematic of the test format for Example 1.

C. results

The Results of this study show that after the hybridization HPV-16 target probes a positive signal detection in CaSki cells (<figref idrefs="S39">2A</figref>) and in SiHa cells (<figref idrefs="S39">2 B</figref>) was observed. As expected, was a Plurality of signals in the CaSki cells is detected, as they approximately 400-600 copies of HPV-16 contain. Only one or two signals were detected in the SiHa cells, also as expected, as they are only 1-2 copies of DNA contain HPV 16th After all no signal in the HPV-16 probes in those cells was detected, which HPV-16 is missing, ie HeLa (<figref idrefs="S39">2C</figref>) and C33a cells (<figref idrefs="S39">2D</figref>). These results both the quantitative capability and the specificity of the present process.

After hybridization with HPV-18 target probes, a positive detection signal in HeLa cells (<figref idrefs="S39">2G</figref>) Observed that 10-50 DNA copies HPV-18 included. However, there was no signal at HPV-18 probe detected in cells lacking HPV-18, including CaSki (<figref idrefs="S39">2E</figref>) SiHa (<figref idrefs="S39">2F</figref>) And C33a cells (<figref idrefs="S39">2H</figref>). There was no signal at either HPV-16 or HPV-18 target probes detected in the HPV-negative HT3 cell line or the ME180 cell line, a DNA sequence carry within them that is similar to that of HPV-39 (not shown).

It were a number of additional Checks performed, to show that the observed in these experiments, positive Signals specific for HPV DNA targets were. There was no positive signal was observed when non-specific Target probes were used, or HPV-16 or HPV-18 target probes as, Amplifier or AP-conjugated label probes from the bDNA ISH method have been omitted. The omission of the Proteinase K digestion or -denaturation of the DNA or the treatment of cells with DNase also resulted in a loss of the signal. As a further control for the DNase digestion experiments the RNase treatment step was omitted to confirm that This loss of signal not to DNase degradation of oligonucleotide probes due was. Under these conditions, a positive signal Sig is detected again, which indicated that the DNA oligonucleotide probes not degraded were and therefore were able to bind to HPV RNA targets.

EXAMPLE 2

AVERAGE SPECIFICITY IN CELLS

It Mixed cell populations were used to determine the specificity of the present Procedure for HPV DNA detection to investigate further. passed Mixed cell samples of unlabeled CaSki cells (containing 400-600 DNA copies of HPV-16) and labeled HeLa cells (containing 10-50 DNA copies HPV-18). The HeLa cells were labeled with the fluorescent cell tracer CFDA SE (Carboxyfluoresceindiacetatsuccinimidylester, Molecular Probes, Eugene, OR) according to the manufacturer's instructions marked. Both cell types were prepared according to the procedure of Example 1 tested.

As in <figref idrefs="S40">3A</figref> and <figref idrefs="S40">3C</figref> shown, showed hybridization with HPV-16 target probes, a positive detection signal only in HPV-16-infected CaSki cells (arrow) and not in HeLa cells (Arrow). Similar, as in the <figref idrefs="S40">3B</figref> and <figref idrefs="S40">3D</figref> shown, showed hybridization with HPV-18 target probes, a positive detection signal only in HPV-18-infected HeLa cells (arrowhead) and not in CaSki cells (arrowhead). As &lt;?page 14?&gt;expected was a stronger signal intensity (Ie greater number and size of the spots) for HPV-16 DNA detection observed in CaSki cells compared to HPV-18 DNA detection in HeLa cells. reflects this difference in signal intensity the higher Number of HPV DNA copies again present in CaSki cells (400-600 HPV-16 DNA copies / cell) as compared to HeLa cells (10-50 HPV-18 DNA copies / cell).

These Results show that the present method in a mixed Population of cells between cells that are infected with HPV-16, and cells infected with HPV-18 DNA, can differ. Furthermore, there is no transfer of the signal from one cell type to another, as positive signals only within the appropriate Cell types are obtained. Furthermore, even a small amount slightly Tar gets with the present method without the problems of the background or cross-reactivity can be detected by the the presence of another HPV genotype could be introduced.

EXAMPLE 3

DETECTION OF HPV RNA AND ISOLATION OF RNA / DNA WITHIN A CELL

For RNA detection cells were grown on chamber slides (no. 12-565-18, Fisher Scientific, Pittsburgh, PA), with 4% formaldehyde in PBS 30 Fixed minutes at RT, with 10 ug / ml proteinase K in PBS for 10 Minutes treated at RT and then twice for 5 minutes washed in PBS. Samples were 3 hours pmol at 40 ° C with 1 HPV-specific target probes in a target probe buffer (6 × SSC, 25 % Formamide, 0.2% BRIJ^® incubated for 35, 0.2% casein) and then according to the same descending series of SSC buffers as described in Example 1 was washed. Similarly Fashion were the Preamplifier- that Amplifier- and AP-conjugated Probe hybridization and washing conditions are the same as described in Example 1st

For subcellular localization of DNA targets, HeLa cells were grown overnight on poly-D-lysine-coated Chamber slides bred. Poly-D-lysine is available from Sigma (Product Code P7280). The next Day, the cell culture medium was removed; the cells were washed with PBS washed and then fixed with 4% formaldehyde in PBS for 30 minutes at RT and to DNA targets by hybridization with HPV-18 target probes or, as negative control, HPV-16 target probes examined.

AP substrate (Fast Red, DAKO Corporation) was added and the slides were incubated for 4 minutes at RT. After terminating the reaction by washing in PBS, the samples were fixed in 4% formaldehyde in PBS 5 Minutes postfixed at RT and then for 40 seconds with either hematoxylin or Bisbenzimid counterstained. Slides were examined, and the images were generated and printed as described in Example 1st

The Hybridization of HeLa cells (prepared for RNA detection) with HPV-18 target probes led to the detection of HPV-18 mRNA, mainly in the cytoplasm (arrowhead, <figref idrefs="S41">4A</figref>). However, it was observed a signal in the same cells, the were incubated with HPV-16 target probes (<figref idrefs="S41">4B</figref>).

in the led contrast the hybridization of HeLa cells (prepared for RNA detection) with HPV-18 target probes to the detection of HPV-18 DNA in HeLa Zellnuclei (arrow, <figref idrefs="S41">4C</figref>). However, it was observed a signal in the same cells, the were incubated with HPV-16 target probes (<figref idrefs="S41">4D</figref>).

These results show that HPV mRNA and HPV DNA to various components are localized in cells. In particular, viral mRNA is principally locates the cytoplasm, whereas viral DNA to the nucleus limited is. These results also show that the positive signals in the compartment of the cell be maintained in the localized the target nucleic acid is. In other words, the target and signal are co-localized.

the This method provides a precise subcellular localization ready resulting positive signals in the subcellular compartments be maintained in which the target nucleic acid sequences are localized.

&lt;?page 15?&gt;

EXAMPLE 4

DETECTION OF HPV-16 BY BDNA ISH IN CIN II SAMPLES

A Protocol, which is similar to that of Example 1, was as follows for the in situ hybridization on tissue used. Briefly, formalin-fixed paraffin sections of cervical tissue and dewaxed using standard histology rehydrated with xylenes and graded alcohol series. For the DNA detection was the fabric at 100 ug / ml RNase in an RNase buffer (0.5 NaCl, 10 mM Tris, pH 9.0, 1 mM EDTA) at 37 ° C for 1 hour long Rnase-digested, followed by Proteinase K digestion (12 ug / ml in Proteinase K buffer or PBS) at 37 ° C 10 minutes. Proteinase K was by 10 minutes post-fixed in 4% Paraformaldehyde in PBS at 4 ° C disabled. After several wash steps in PBS the tissue was acetylated in 1 M TEA as described by Angerer et al., In situ Hybridization with RNA Probes An Annotated Recipe, In: KL Valentine et al. (Ed.), In situ Hybridization-Applications to Neurobiology, 71-96, Oxford, Oxford University Press (1987). Sections were in dehydrated ethanol series before for 5 minutes at 92 ° C in DB (80 % Formamide, 2 x SSC) were denatured on Hybaid (Phenix), followed by immersion in cold 70% ethanol and dehydration. After a 30 minute incubation in a prehybridization buffer were target oligonucleotide in fresh prehybridization buffer added at a concentration of 10 fmol / ul and covered with a cover glass.

The Hybridization was performed in a humidified chamber on a Fisher slide warmer at 37 ° C 1-3 Hour (s) carried out for with washes and Signalamplifiaktion, which were the same, as previously described in Example 1st After hybridization, were cuts in a graded SSC series to 0.1 × SSC for a total period of time washed 15 minutes wash time. Hybridization in 1 fmol / ul Preamplifier in hybridization solution B for 25 minutes at 55 ° C was by 3 washes in 0.1 × SSC at 5 minutes of total washing time followed. Hybridization in 1 fmol / ul Amplifier in hybridization solution B 25 Minutes at 55 ° C was supported by several 0.1 × -SSC washes followed. Hybridization in 1 fmol / ul label probe in Hybridization Solution B 15 Minutes at 55 ° C was of 0.1 × -SSC washes and incubation in wash solution D followed. Fast Red was immediately prior to application to the manufactured sections, and color development was between 4 and stopped for 10 minutes. Nuclei were stained in hematoxylin and the slides were for Microscopy covered with a cover glass. Endogenous alkaline Phosphatase was disabled with levamisole, 1 uM to 5 uM.

The In situ hybridization detected HPV-16 in CIN II uterine cervical tissue (CIN = intraepithelial cervical neoplasia) with HPV-associated cytopathic changes (Sample Nos. 00B01-627, Clinomics BioSciences, Inc., Pittsfield, MA). The Zellnuclei in each of the sections were stained with hematoxylin colored. <figref idrefs="S42">5A</figref> provides the distribution of HPV-16 gene expression (stained areas) within Schuppenepithelen the ectocervix. A higher magnification of einrahmten Region in <figref idrefs="S42">5A</figref> shows the presence of HPV-16 mRNA in the cytosol some squamous epithelial cells (<figref idrefs="S42">5B</figref>). <figref idrefs="S42">5C</figref> shows, that HPV 16 DNA in the nuclei of squamous cells in a tissue section in addition to the boxed region in <figref idrefs="S42">5A</figref> was detected. HPV-18 DNA, however, was not in an adjacent section detected (<figref idrefs="S42">5D</figref>). As expected, GAPDH mRNA throughout the squamous epithelia near the transformation zone detected (Fig. E). The<figref idrefs="S42">5A</figref> and <figref idrefs="S42">5E</figref> are 200 × magnified, while the <figref idrefs="S42">5B</figref>. <figref idrefs="S42">5C</figref> and <figref idrefs="S42">5D</figref> × 600 are enlarged.

EXAMPLE 5

BDNA ISH OF HPV DNA WITH RETENTION THE hISTOPATHOLOGY

The In-situ hybridization using the method that similar to that of Example 4, was carried out, to HPV-16 DNA to be detected in regions of cytopathic changes, the for CIN II lesions in Uterine cervical tissue with HPV-associated cytopathic changes are characteristic (Sample Nos. 00B01-624, Clinomics BioSciences, Inc., Pittsfield, MA), as follows. The Zellnuclei in were sections with hematoxylin stained. Size Arrowheads indicate contiguous along Basaizellen the basement membrane through, located the the squamous epithelium from including Stream separates. In a region of seemingly normal maturation shed HPV-16 DNA was not detected, basal cells were in the close Basement membrane regions, and the apical part of the epithelium comprised mainly differentiated Squamous cells with a characteristically large cytosol (<figref idrefs="S43">6A</figref>). A nearby region of the same tissue section showed a abnormal shed maturation, for moderate dysplasia (CIN II) is characteristic (<figref idrefs="S43">6B</figref>). This dysplastic region exhibited overgrowth of Basal cells, mixing of and the differentiated Basaizellen Squamous cells in the apical Epi&lt;?page 16?&gt;thelregion and the presence of HPV-16 DNA (stained Area) (<figref idrefs="S43">6B</figref>). Higher magnification showed the presence HPV-16 DNA in basal cells, as shown by the smaller arrows becomes (<figref idrefs="S43">6C</figref>). The<figref idrefs="S43">6A</figref> and <figref idrefs="S43">6B</figref> are 400 × increased, and <figref idrefs="S43">6C</figref> is 600 × magnified.

EXAMPLE 6

STUDY OF SPECIFICITY IN TISSUE

The In situ hybridization using genotype-specific HPV probes and the process of Example 4 were used to detect HPV DNA in CIN II tissue sections and to detect in normal Zervixschnitten. The Zellnuclei in the sections were counterstained with hematoxylin. The colored Areas indicated the presence of the target nucleic acid, either viral DNA (<figref idrefs="S44">7A</figref>. <figref idrefs="S44">7B</figref>. <figref idrefs="S44">7C</figref>. <figref idrefs="S44">7D</figref>. <figref idrefs="S44">7E</figref> and <figref idrefs="S44">7F</figref>) or endogenous mRNA (<figref idrefs="S44">7G</figref>). HPV-16 DNA was in ectocervical Schuppenepithelen of CIN II tissue Nr. 00B01-624 (<figref idrefs="S44">7A</figref>) detected HPV-18 was not detected (<figref idrefs="S44">7B</figref>). HPV 18 DNA was in CIN II tissue with HPV-associated cytopathic changes (sample no. 00B01-625, Clinomics BioSciences, Inc., Pittsfield, MA) is detected, as shown in <figref idrefs="S44">7D</figref> shown, HPV-16 was not detected (<figref idrefs="S44">7C</figref>). As expected HPV DNA was not detected in normal cervical tissue (sample No. H-1188-88, Clinomics BioSciences, Inc., Pittsfield, MA), if with HPV-18- (<figref idrefs="S44">7E</figref>) Or HPV-16 probes (<figref idrefs="S44">7F</figref>) was tested. As expected, endogenous GAPDH mRNA in normal Cervical tissue detected (<figref idrefs="S44">7G</figref>).