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

IS 2 287 134 T3

DESCRIPTION

Procedure for the detection and localization of genes in situ using branched DNA hybridization.

Technical field

This invention relates generally to nucleic acid chemistry and biochemical assays. More particularly, the invention relates to highly sensitive methods for the in situ detection of a nucleic acid analyte. The method employs branched DNA oligoprobes and hybridization techniques to detect and identify the location of a nucleic acid analyte within a biological sample.

Previous technique

Branched DNA (rDNA) signal amplification technology has been used extensively in a microwell format to detect and quantify specific nucleic acid sequences. Urdea et al. (2000) Branched-DNA (bDNA) Technology. In Kessler C., ed., Nonradioactive Analysis of Biomolecules, New York, Springer-Verlag: 388-395. Inherently quantitative and highly reproducible rDNA can be applied to the detection of any nucleic acid target for which a sequence is known without the use of radioactive probes.

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

More recently, rDNA technology has been used to detect and measure cellular mRNA expression, including: cytokines, Breen et al. (1997) Cell Immunol 178: 91-98 and Shen et al. (1998) J Immunol Methods 215: 123134; progesterone and estrogen receptors, Nargessi et al. (1998) Breast Cancer Res Treat 50: 47-55 and Nargessi et al. (1998) Breast Cancer Res Treat 50: 57-62; insulin, Wang et al. (1997) Proc Natl Acad Sci USA 94: 43604365; glucokinase, Cabrera-Valladares et al. (1999) Endocrinology 140: 3091-3096; c-fos, Shyamala et al. (1999) Anal Biochem 266: 140-147; and aP2, Burris et al. (1999) Mol Endocrinol 13: 410-417. All of these rDNA assays were developed to measure nucleic acids in serum, plasma, or cell lysates. However, none of these studies suggest or describe an rDNA assay for the detection of nucleic acids in morphologically intact tissues or cells.

In contrast, an in situ hybridization assay (HIS) would necessarily have the ability to detect specific nucleic acid sequences in morphologically intact tissues or cells. HIS procedures have improved since the general concept was introduced by Pardue and Gall more than twenty years ago. Pardue et al. (1969) Proc Natl Acad Sci USA 64: 600-604. For example, the use of non-isotopic probes has eliminated the inherent problems associated with radioactive HIS procedures such as long response times, risks of exposure to radioactivity, and waste disposal. Furthermore, the incorporation of various targets and signal amplification systems has improved the sensitivity of HIS. However, despite these advances in HIS procedures, several challenges still need to be overcome. These challenges include developing sensitive and specific target detection, ensuring precise signal and target co-localization, preserving target sequences, and preserving cell and tissue morphology. In addition, the HIS procedures must address several practical issues, including ease of use, reproducibility, and quantifiability, automation susceptibility, versatility, and timely completion.

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

A study has shown that it is possible to adapt rDNA technology to a HIS format for the detection of mRNA. Cao et al. (1998) Proceedings of the American Association for Cancer Research, 89^to Reunion, New Orleans, LA and Antao et al. (1999) In Situ Hybridization Using the bDNA Technology. In Patterson, BK ed., Techniques in Quantification and Localization of Gene Expression, Boston, Birkhauser Press: 81-93. Antao et al. describe the use of rDNA technology for in situ hybridization to RNA in individual cells. The main steps in the rDNA assay are prehybridization, sequential hybridization with target probes, preamplifier probe, amplifier and marker, and finally the generation and detection of signals. However, the assay described in this study has a relatively low sensitivity.

Thus, there remains a need in the art to provide highly sensitive methods for the in situ detection of nucleic acid analytes in biological samples. The present invention satisfies these and other needs in the art.

IS 2 287 134 T3

Description of the invention

Accordingly, it is a primary objective of the invention to address the needs described above in the art by providing a method for the in situ detection of a nucleic acid analyte within a sample of biological material based on rDNA hybridization, wherein the procedure results in increased sensitivity.

The method of the invention can be used to identify the position, ie subcellular location, of a nucleic acid analyte within a single cell based on an rDNA hybridization technique.

Additional objects, advantages, and novel features of the invention will be set forth in part in the following description, and in part will be apparent to those skilled in the art upon examining the following, or may be learned by practice of the invention.

According to the invention, therefore, there is provided a method for the in situ detection of a nucleic acid analyte within a sample of biological material that comprises performing rDNA hybridization comprising the steps of:

(a) prepare a sample of biological material:

(i) immobilizing the biological material on a substrate;

(ii) permeabilizing the biological material bound to the substrate by contacting the biological material bound to the substrate with a solution containing Proteinase K at a concentration of 0.5 pg / ml to 50 pg / ml;

(iii) treating the sample with RNase so that all RNA in the sample is removed; and (iv) heating the permeabilized biological material to a temperature and for a period of time effective to denature any double-stranded DNA;

(b) contacting the biological material with a target oligonucleotide probe under hybridization conditions, in which at least a part of the target probe is complementary to at least a part of the nucleic acid analyte, so that the analyte-target probe complex when the nucleic acid analyte is present in the sample;

(c) washing the biological material with a washing fluid comprising a detergent, at a temperature in the range of 21 ° C to 60 ° C; and (d) detecting any analyte-target probe complex on the substrate (i) by contacting the washed substrate and analyte-target probe complex with a preamplifier oligonucleotide probe under hybridization conditions, wherein a first part of the preamplifier probe is complementary to a part of the target probe other than the part of the target probe that is complementary to the nucleic acid analyte, thus forming an analyte-preamplifier target probe complex when the nucleic acid analyte is present in the sample;

(ii) contacting the product of step (d) (i) with an oligonucleotide amplifier probe under hybridization conditions, in which a first part of the amplifier probe is complementary to a second part of the preamplifier probe , thus forming an analyte-preamplifier target probe-amplifier probe complex when the nucleic acid analyte is present in the sample;

(iii) contacting the product of step (d) (ii) with a label probe comprising an alkaline phosphatase-conjugated oligonucleotide probe under hybridization conditions, wherein a part of the label probe binds to a second part of the amplifier probe, thus forming an analyte-target probe-preamplifier probe-amplifier probe-marker probe complex when the nucleic acid analyte is present in the sample;

(iv) labeling the analyte-target probe-preamplifier probe-amplifier probe-marker probe complex with a detectable marker; and (v) detecting the presence of the marker on the substrate, wherein the nucleic acid analyte is DNA, an endogenous gene, or a segment thereof.

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

IS 2 287 134 T3

In a second embodiment, a method is provided for identifying the position of a nucleic acid analyte within a cell from a sample of biological material based on rDNA hybridization. This procedure for locating and identifying the position of a nucleic acid analyte comprises the same steps as those described above with respect to the in situ detection of a nucleic acid analyte. However, once the marker is detected, identification of the position of the target probe-analyte complex within a cell of the biological sample is indicative of the position of the nucleic acid analyte within the cell.

Brief description of the drawings

Figure 1 is a schematic representation of an rDNA HIS procedure according to the present invention.

In another embodiment, a method is provided for detecting a nucleic acid analyte within a sample of biological material, the method comprises performing rDNA hybridization to detect the nucleic acid analyte in situ, wherein the method has sufficient sensitivity. to detect from about 1 copy to about 10 copies of the nucleic acid analyte in the biological material.

Brief description of the drawings

Figure 1 is a schematic representation of an rDNA HIS procedure according to the present invention.

Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H are microscopic images (60X) of the results obtained in Example 1.

Figures 3A, 3B, 3C and 3D are microscopic images (60X) of the results obtained in Example 2.

Figures 4A, 4B, 4C and 4D are microscopic images (60X) of the results obtained in Example 3.

Figures 5A, 5B, 5C, 4D and 5E are microscopic images of the results obtained in Example 4.

Figures 6A, 6B and 6C are microscopic images of the results obtained in Example 5.

Figures 7A, 7B, 7C, 7D, 7E, 7F and 7G are microscopic images of the results obtained in Example 6. Modes for carrying out the invention I. Definitions and summary

Before describing the present invention in detail, it should be understood that this invention is not limited to specific probes, reagents, assay formats, or the like, as they may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It should be noted that, as used in this specification and appended claims, the singular forms "a", "an", "the" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an analyte-target probe complex" includes two or more such complexes, reference to a "wash" step includes two or more such steps, and the like.

In this specification and the claims that follow, the following terminology will be used in accordance with the definitions set forth below.

"Oligonucleotide" shall be 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 that be it an N-glucoside of a purine or pyrimidine base, or of a modified purine or pyrimidine base. The oligonucleotides can be single-stranded or double-stranded, usually single-stranded. Furthermore, the oligonucleotides used in the present invention usually have from about 2 monomer units to about 100 monomer units, more usually from about 2 monomer units to about 80 monomer units, and most usually from about 2 monomer units to about 60 monomer units.

The term "nucleic acid analyte" refers to a single-stranded or double-stranded nucleic acid molecule that contains a target nucleotide sequence. The nucleic acid analyte can be from a variety of sources, eg, biological solids or liquids, food products, environmental materials, etc. The term "nucleic acid analyte" is used interchangeably herein with the term "analyte."

As used herein, the terms "target region" or "target nucleotide sequence" refer to a probe binding region contained within the nucleic acid analyte. The target region must be at least 400 bases in length. The term "target sequence" refers to a sequence with which a probe, ie, a target oligonucleotide probe, will form a stable hybrid under the desired conditions.

IS 2 287 134 T3

As used herein, the terms "probe" and "oligonucleotide probe" refer to a structure comprised of an oligonucleotide as defined above that contains a nucleic acid sequence complementary to a portion of a target nucleotide sequence. , at least one other probe, or both. The oligonucleotide regions of the probes can be composed of DNA, and / or RNA, and / or synthetic nucleotide analogs.

It will be appreciated that the linking sequences need not have perfect complementarity to provide stable hybrids. In many situations, stable hybrids will form when less than about 10% of the bases are mismatches, ignoring loops of four or more nucleotides. Consequently, the term "complementary" refers to an oligonucleotide that forms a stable duplex with its "complement" under the conditions of the assay, generally when there is a homology of about 90% or greater.

As used herein, the terms " biological sample" or "biological material" are used interchangeably and each refers to a tissue, cell, or fluid sample isolated from an individual, including, but not limited to to, for example, plasma, serum, cerebrospinal fluid, semen, lymphatic fluid, the outer sections of the skin, respiratory tract secretions, intestinal tract secretions, genitourinary tract secretions, tears, saliva, milk, blood cells, tumors, organs, and also in vitro cell culture constituent samples (including, but not limited to, conditioned medium resulting from the growth of cells in cell culture medium, putative virus-infected cells, recombinant cells , and cellular components). It is preferred that the biological sample is in the form of a liquid, eg, tissue or cells in a liquid, although solid tissue can also be used. The preferred uses of the present method are the detection and / or quantification of nucleic acids as follows: (a) viral nucleic acids, such as hepatitis B virus ("HBV"), hepatitis C virus ("HCV" ), hepatitis G virus (“VHG”), human immunodeficiency virus (“HIV”), human papillomavirus (“HPV”), and the herpes family of viruses, including herpes zoster (chickenpox), virus herpes simplex types I and II, cytomegalovirus ("CMV"), and Epstein-Barr virus; (b) bacterial nucleic acids, such as chlamydia, Mycobacterium tuberculosis, etc .; and (c) numerous human sequences of interest, including lysosomal acid phosphatase ("LAP"), interleukin-2 (IL-2), and interferon-gamma (INF).

The term "hybridization conditions" is intended to mean those conditions of time, temperature and pH and the necessary amounts and concentrations of reactants and reagents sufficient to allow at least a portion of a probe to anneal with its complementary sequence. As is well known in the art, the time, temperature, and pH conditions required to achieve hybridization depend on the size of the oligonucleotide probe to be hybridized, the degree of complementarity between the oligonucleotide probe and the target, and the presence of other materials in the hybridization reaction mixture. The actual conditions required for each hybridization step are well known in the art or can be determined without undue experimentation.

Typical hybridization conditions include the use of solutions buffered to a pH of from about 7 to about 8.5 and are carried out at temperatures of from about 30 ° C to about 55 ° C, preferably from about 37 ° C to about 55 ° C. C for a period of time from about 1 second to about 1 day, preferably from about 15 minutes to about 16 hours, and most preferably from about 15 minutes to about 3 hours.

The "hybridization conditions" also require an effective buffer. Any buffer can be used that is compatible, ie, chemically inert, with respect to probes and other components, but still allows hybridization between complementary base pairs. A particularly preferred buffer, hereinafter referred to as "hybridization solution A", comprises 3X SSC, 50% formamide, 10% dextran sulfate (MW 500,000), 0.2% casein, 10 mg polyA. / ml, 100 ng / ml denatured salmon sperm DNA in which 1X SSC is 0.15 M sodium chloride and 0.015 M sodium citrate. Another particularly preferred buffer, hereinafter referred to as "Hybridization Solution B", comprises 5X SSC, 0.1-0.3% sodium dodecyl sulfate, 10% dextran sulfate, ZnCl₂ 1 mM, and MgCl₂ 10 mM where 1X SSC is as defined above.

"Optional" or "optionally" means that the circumstance described below may or may not occur, so that the description includes cases in which the circumstance does occur and cases in which it does not. For example, "optionally heating the permeabilized biological material" includes cases where the permeabilized biological material is heated and cases where the permeabilized biological material is not heated.

The method of the present invention is an in situ rDNA hybridization assay. As appreciated in the art, rDNA-based methods effectively provide for amplification of a signal that may not otherwise be detectable with other techniques. Those skilled in the art are familiar with the techniques and materials necessary to perform rDNA-based assays. Briefly, in rDNA amplification, a target oligonucleotide probe comprises two sites: one to specifically hybridize to a part of the nucleic acid analyte, and one to specifically hybridize to at least one other probe. Additional oligonucleotide probes with unique sites designed to specifically hybridize to different probes can be used, such that as repeated phases of hybridization occur, a branched DNA structure is formed. The final oligonucleotide probe to be annealed into the branched structure carries at least one detectable label. Thus, the original target molecule gives rise to a multitude of signals, thus amplifying the signal for easy detection. In addition, you can

ES 2 287 134 T3 reference is made to the relevant literature, texts and other references for a description of conventional rDNA techniques. See, for example, Collins et al. (1997) Nucleic Acid Res. 25 (15): 2979-2984.

The present invention provides highly sensitive techniques for detecting a nucleic acid analyte based on in situ rDNA hybridization. Previous in situ rDNA hybridization techniques have provided relatively non-sensitive results. However, the present invention provides an in situ rDNA method that is highly sensitive, eg, having sufficient sensitivity to detect from about 1 to about 10 copies of a nucleic acid analyte in a biological material.

II. The screening procedure

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

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

The sample of biological material is obtained by conventional procedures and includes, for example, biopsy and extraction of biological fluids. However, since the technique is for in situ analysis, tissues, cells or organelles are analyzed rather than a lysate. Care must be taken in obtaining and handling the tissue, cell, or organelle throughout the assay to ensure that the material remains substantially intact.

The biological sample for use in the present procedure, that is, the tissues or cells to be analyzed, can be taken from any part of the body as long as the biological material is suspected of containing the analyte of interest. For example, tissues suitable for use in the present method include, without limitation, adrenal, bladder, bone marrow, brain, breast, cardiac, colon, esophagus, intestinal, kidney, liver, lung, lymph node, nerve, tissue, ovaries, pancreatic, prostate, skeletal (striated) muscle, smooth muscle, spleen, stomach, testes, tonsils, tracheal and uterine. Furthermore, cells taken from these same tissues are suitable for the techniques described herein. Additionally, cells can be obtained from cell-containing fluids such as plasma, serum, cerebrospinal fluid, etc., as explained above. Initially, the biological material must be conserved or “fixed”. Although many procedures are known in the art for fixing biological samples, it is preferred that the biological sample is fixed with formaldehyde. For example, the biological material can be combined with a 4% formaldehyde solution for 30 minutes on ice. Alternatively, other fixing agents, for example alcohol, can also be used. Once fixed, the biological material must be immobilized on a substrate. Suitable substrates are comprised of those materials that allow immobilization of a tissue, cell, or organelle while preserving the morphology of the sample. The substrate material must also be solid in addition to being heat resistant. Furthermore, the substrate must be inert with respect to the reagents used. Preferred substrates comprise glass, for example a glass slide, although an elastic plastic can also be used.

The biological material can be immobilized on the substrate using standard procedures. For cells, it is preferred that the biological material is immobilized using a centrifuge. Generally, the force required to immobilize the biological material on the substrate is from about 200 xg to about 500 xg (times the force of gravity). It is preferred that the force used is about 300 x g.

Immobilization of tissue samples is preferably carried out by cutting very thin sections of the tissue sample and placing them on a suitable substrate. Preferably, the tissue is first placed in a cryostat and cooled to about -15 ° C to about -20 ° C in order to freeze the tissue. Then a microtome is used to cut the frozen tissue into sections. Subsequently, a single section is placed on a substrate, eg glass slides, and the section is allowed to "melt" onto the substrate, thereby immobilizing the sample. Wax infused tissues, eg paraffin, can be used in place of frozen tissues. As will be appreciated, other procedures can be used to immobilize the tissue sample.

In order to ensure that the oligonucleotide probes have access to the internal medium, it is necessary to make the biological material bound to the substrate permeable. It has been found that contacting the substrate-bound biological material with a solution containing proteinase K at a concentration of from about 0.5 pg / ml to about 50 pg / ml, preferably from about 5 pg / ml to about 20 pg / ml (from a stock solution having an activity of 600 units per ml), makes the biological material permeable to a sufficient degree such that the probes pass freely through the biological membranes while maintaining the morphology of the sample. The time and temperature parameters to make the biological material permeable are well known or can be determined experimentally. A reaction time of about 10 minutes at about 37 ° C is preferred to permeabilize with proteinase K.

When the nucleic acid analyte comprises double-stranded DNA, it is necessary to denature the DNA in such a way that hybridization with probes can take place by exposing the sample to heat treatment of a temperature and for a period of time effective to denature any stranded DNA. double. However, heating of DNA from about 1.5 minutes to about 5 minutes has been found to

ES 2 287 134 T3 nutes is sufficient to denature double-stranded DNA while preserving cellular components. The temperature can be any temperature known in the art sufficient to denature DNA. However, it is preferred that the temperature is from about 70 ° C to about 92 ° C, preferably from about 80 ° C to about 92 ° C, with a temperature of about 92 ° C being most preferred.

Furthermore, when the nucleic acid analyte comprises DNA, it is also necessary to digest any RNA that may be present. By digesting RNA, the probes cannot hybridize with RNA, thus minimizing possible false positive results. Generally, any method known in the art can be used to digest RNA. However, it is preferred to use RNase (available from Sigma, Fluka, etc.). RNase can be added to the sample in an amount from about 25 pg to about 100 pg (per spot on the slide) and allowed to incubate for a period of time and at a temperature sufficient to digest the RNA. However, it is preferred that approximately 40 pg (per spot on the slide) of RNase is added to the sample and held at 37 ° C for one hour.

Other procedures are available to prepare the biological material. For example, using commercially available kits such as the THINPREP® slide system (Cytyc Corporation, Boxborough, MA).

Once prepared, the biological material is placed in contact with a target oligonucleotide probe under hybridization conditions. The target probe has a part that is complementary to at least a part of the target sequence of the nucleic acid analyte. When the nucleic acid analyte of interest is present in the sample, the nucleic acid analyte and the target probe hybridize to form an analyte-target probe complex.

The amount of target probes added can be determined experimentally, but it is preferred that from about 0.1 pmol to about 10 pmol (per spot on the slide) are added and allowed to incubate for about three hours at 40 ° C. A preferred reaction medium for this hybridization step is Hybridization Solution A (defined above).

After sufficient incubation time has elapsed, both the substrate and the target analyte probe complex, if present, are washed to facilitate removal of unbound target probes. The washing step requires the use of a washing fluid which generally comprises a detergent and generally a buffer solution.

The buffer solution can be any conventional solution known in the art suitable for removing unhybridized oligonucleotide probes. Preferred buffer solutions comprise the alkali metal salts. Particularly preferred buffer solutions comprise sodium chloride, sodium citrate, and combinations thereof.

The detergent is preferably a non-ionic detergent. Furthermore, it is preferred that the detergent is also a hydrophilic surfactant. Exemplary detergents are polyoxyethylene-based detergents, eg BRIJ® and TRITON®. Similar detergents also suitable for use in the present invention are sold under the trade names TWEEN®, GENAPOL®, IGEPAL CA®, THESlT®, and LUPROL® (all available from commercial suppliers).

After determining the washing fluid, the washing step is carried out at least one, preferably two, and most preferably three times. The temperature of the wash step has been found to influence the sensitivity of the assay. Therefore, for the present process, temperatures in the range of about 21 ° C to about 60 ° C work well and are used. Optimally, the washing step is carried out at room temperature.

Upon completion of the wash step, the non-hybridized target probes are absent. Analyte-target probe complexes are then detected on the substrate to determine the presence of the nucleic acid analyte. Consequently, additional oligonucleotide probes are added in such a way that a branched network is formed. Once a branched network is formed, a plurality of detectable markers are added with the effect of "amplifying" the signal for easy detection. Therefore, the detection of a target analyte-probe complex is achieved by:

(d) (i) contacting the washed substrate and analyte-target probe complex with a preamplifier oligonucleotide probe under hybridization conditions, in which a first part of the preamplifier probe is complementary to a different target probe part of the part of the target probe that is complementary to the nucleic acid analyte, thus forming an analyte-target probe-preamplifier probe complex when the nucleic acid analyte is present in the sample;

(d) (ii) contacting the product of step (d) (i) with an amplifier oligonucleotide probe under hybridization conditions, in which a first part of the amplifier probe is complementary to a second part of the preamplifier probe, thus forming an analyte-target probe-preamplifier probe-amplifier probe complex when the nucleic acid analyte is present in the sample;

(d) (iii) contacting the product of step (d) (ii) with a label probe comprising an oligonucleotide probe conjugated to alkaline phosphatase under hybridization conditions, in which a part of the probe of

ES 2 287 134 T3 marker binds to a second part of the amplifier probe, thus forming an analyte-target probe-preamplifier probe-amplifier probe-marker probe complex when the nucleic acid analyte is present in the sample;

(d) (iv) labeling the analyte-target probe-preamplifier probe-amplifier probe-marker probe complex with a detectable label; and (d) (v) detecting the presence of the marker on the substrate.

Each of the probe hybridization steps, i.e., steps (d) (i), (d) (ii) and (d) (iii), is carried out under standard hybridization conditions as discussed above. . However, for these particular hybridization steps it is preferred that each step take place at about 55 ° C with an incubation time of about 25 minutes. It is also preferred that each probe, ie, preamplifier probe, amplifier probe, and marker probe, is separately mixed with Hybridization Solution B (defined above) to contact that probe with the growing complex. Thus, for example, the amplifier probe is mixed with 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 probes added can be determined using techniques known in the art, it is preferred that the preamplifier probe and amplifier probe are each added in an amount of from about 1 fmol to about 10 pmol (per spot on the slide). Most preferably, each of these probes are added in an amount of from about 1 fmol to about 100 fmoles (per spot on the slide).

Labeling is achieved when the marker probe hybridizes to the analyte-target probe-preamplifier probe-amplifier probe complex. The marker probe includes one or more detectable markers that directly or indirectly provide a detectable signal. The markers can be attached, covalently or non-covalently, to the marker probe as individual elements of the complementary sequence, or they can be present as a terminal element or terminal tail having a plurality of markers. Various means of providing markers linked to a probe have been reported in the literature. See, for example, Leary et al. (1983) Proc Natl Acad Sci USA 80: 4045; Renz et al. (1984) Nucl Acids Res 12: 3435; Richardson et al. (1983) Nucl Acids Res 11: 6167; Smith et al. Nucl Acids Res (1985) 13: 2399; Meinkoth et al. (1984) Anal Biochem 138: 267.

Labels that may be employed include agents that can fluoresce, agents that can chemiluminescer, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and the like. Illustrative specific markers include fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, α-β-galactosidase, horseradish peroxidase, and alkaline phosphatase, among others. At least one alkaline phosphatase marker is used.

Detection of the detectable marker can be accomplished by any means known in the art and is dependent on the nature of the marker. For agents that can fluoresce, there are a large number of fluorometers available. For agents that can chemiluminescer, there are luminometers or films available. With the enzymes, a fluorescent, chemiluminescent or colored product can be provided and determined fluorometrically, luminometrically, spectrophotometrically or visually (preferably with the aid of a microscope). For the present procedure, it is preferred that the alkaline phosphatase substrate is added to detect the presence of the alkaline phosphatase marker using fluorescence or bright field microscopy.

Contrary to previous assays, the present method for detecting a nucleic acid analyte is highly sensitive. Previous assays were based on the presence of many copies (often hundreds) of the nucleic acid analyte. The present method has been developed to detect relatively few copies. Therefore, it is preferred that the method has a sensitivity sufficient to detect from 1 copy to about 10 copies of the nucleic acid analyte. However, it is most preferred that the method has sufficient sensitivity to detect from 1 copy to about 2 copies of the nucleic acid analyte.

III. Probe synthesis

The sequences of the probes are determined using standard techniques known in the art. Thus, for example, the target probe sequence will be determined using a known analyte sequence of interest specific for that analyte. Those regions of the sequences that are intended to be involved in binding (and thus are complementary to another oligonucleotide sequence, be it a probe or analyte) will each have at least 15 nucleotides, usually at least 20 nucleotides, and no more. of approximately 100 nucleotides. Typically, the binding sequences will be about 25 nucleotides in length. They will normally be chosen to bind to different sequences of the analyte and / or to different and specific parts of the various probes. Furthermore, the oligonucleotides for any given set of probes will optimally have the same melting temperature.

Probes with a second binding sequence are selected to be substantially complementary to the appropriate part of the probe. The second linking sequence may be contiguous to the first linking sequence, or it may be separated from it by a non-complementary intervening sequence. Probes may include other

ES 2 287 134 T3 non-complementary sequences if desired. However, these non-complementary sequences should not hinder the binding of the binding sequences or result in non-specific binding.

Probes can be prepared by oligonucleotide synthesis or cloning, the former being preferred. As is well known in the art, procedures for synthesizing oligonucleotides typically involve the sequential addition of 3 'blocked and 5' blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing oligonucleotide chain, in which each addition is done by nucleophilic attack of the terminal 5'hydroxyl group of the growing chain on the 3 'position of the added monomer which is usually a phosphorus derivative such as phosphotriester, phosphoramidite, or the like.

IV. Location

The present invention also provides a method for determining the location or position of a nucleic acid analyte in a cell. As indicated above, this procedure comprises the same steps as in situ detection but adds an additional step of (e) identifying the position of the analyte-target probe complex within a cell of the biological sample as indicative of the position of the analyte. nucleic acid in the cell. Previous in situ detection assays were less accurate in demonstrating the subcellular position of the signal. For example, the signal from previous in situ assays may not be "contained" within the subcellular compartment. Alternatively, the signal from previous in situ assays could be spread throughout a relatively large part of the cell, thus making a definitive conclusion regarding the location of the nucleic acid analyte impossible. Because the temperatures for hybridization are low, that is, they generally do not exceed 55 ° C, and the sample treatment is not harsh, the present procedure enables the ability to determine the location of a single analyte within a sample. cell. Even when the temperature is increased for certain steps, for example, up to about 92 ° C when DNA is denatured by heating, the present method still retains the ability to determine the location of a single analyte within a cell.

V. Utility

The present method can be used to detect any DNA, endogenous gene or segment thereof for which the sequence is known. Preferred analytes for which the method is particularly well suited include, but are not limited to, HIV DNA; CMV DNA, HPV DNA, LAP, IL-2, and gene transcripts.

Taking into account its sensitivity, ease of use, versatility, reliability and speed (results are obtained within a day), the present procedure has a wide application in the early detection of cancers and infectious diseases. Detection of a single copy is particularly advantageous for viral diseases, eg HIV and many others, since the diagnosis can be achieved much earlier (when viral loads are relatively low), thus allowing earlier intervention and treatment. Furthermore, the present method has applications in the field of treatment selection. For example, the present method can be adapted to rapidly and accurately detect human epidermal growth factor receptor 2 (HER2 / neu) gene copy number. This is useful in deciding whether or not to initiate anti-HER2 treatment, eg, anti-HER2 monoclonal antibody treatment (Herceptin®, available from Genentech, Inc., South San Francisco, CA). Furthermore, by modifying the pretreatment conditions of cells or tissues, the present method can detect either mRNA or DNA with the same set of probes.

The present method is highly specific and allows precise localization of the target sequence within the cell when a part of the nucleic acid analyte sequence is known. Furthermore, localization is possible even when there is only a single copy of the nucleic acid analyte in the cell. Therefore, such an assay has wide application and is particularly useful in research and medicine. In gene therapies, for example, it is now possible to determine whether the drug enters the cytoplasm and / or nucleus.

In addition to detecting cancers and infectious diseases and assisting with treatment selection, the ability to detect nucleic acids within tissue sections using the present methods has additional advantages. For example, in situ detection within a tissue sample demonstrates the extent to which a cancer or infectious pathogen has spread to areas around the tissue. Such information serves as a prognostic indicator and provides the ability to tailor an appropriate therapeutic approach to the disease.

The scope of the invention is defined by the appended claims.

Experimental part

The following examples are set forth so as to provide those of ordinary skill in the art with a full disclosure and description of how to prepare and use the compounds described and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (eg quantities, temperature, etc.), but some errors and deviations must be accounted for. Unless otherwise stated, temperature is expressed in ° C and pressure is at or near atmospheric pressure at sea level.

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Unless otherwise indicated, all starting materials and reagents were obtained commercially (eg, from Aldrich, Sigma, and ICN) and used without further purification. Standard cell culture and cell harvesting procedures were used.

Example 1

Detection of HPV DNA by rDNA HIS in cells

A. Materials and procedures

i. Cell culture

Because they contain different strains and amounts of human papillomavirus (HPV), HeLa, CaSki, and SiHa human cervical carcinoma cell lines were used to evaluate the present invention. Table 1 describes each cell line.

TABLE 1

<td>Cellphone line*</td><td>Viral strain</td><td>DNA copies per cell</td>

<td>CaSki</td><td>Integrated HPV-16 DNA</td><td>400-600 copies</td>

<td>HeLa</td><td>Integrated HPV-18 DNA</td><td>10-50 couplets</td>

<td>Yes</td><td>Integrated HPV-16 DNA</td><td>1-2 couplets</td>

<td>C33A (control)</td><td>Negative for HPV</td><td>0 couplets</td>

* All cell lines were obtained from the American Type Cell Culture Collection (ATCC; Manassas, VA) and grown in flasks under conditions generally suggested by the ATCC. When required, cells were removed from the flasks using a gentle trypsin treatment.

ii. Oligonucleotide probes

The HPV-16 specific target probes consisted of a total of 26 DNA oligonucleotide probes covering approximately 90% of the E6 and E7 regions of the HPV genome. These probes were designed by first creating a degenerate consensus sequence based on 23 sequences for the E6 and E7 genes of the HPV-16 genome, retrieved from GenBank using Genetics Data Environment software (Harvard Genome Laboratory, Cambridge, MA). Sets of possible target probes were then generated using ProbeDesigner® software (Bayer Diagnostics, Emeryville, CA), which allows flexible design of rDNA probe sets with minimal contribution to background and designates a constant melting temperature of 63 ± 2. ° C. Final probes were selected after scanning the probe sets for possible interactions with the other 39 HPV genotypes using HybSimulator.^® (Advanced Gene Computing Technologies, Inc., Irvine, CA) and Human Genomic DNA Sequences Using Blast2^TM (National Institutes of Health, Bethesda, MD).

The HPV-18 specific target probes consisted of a total of 32 DNA oligonucleotide probes that covered 90% of the E6 and E7 regions of the HPV genome. These probes were designed 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.

Other DNA oligonucleotide probes have been described in detail, including preamplifier, amplifier, and alkaline phosphatase (FA) conjugated marker probes. Collins et al. (1997) Nucleic Acids Res 25: 2979-2984. To reduce non-specific hybridization, the unnatural nucleotides 5-methyl-2'-deoxyisocytidine (isoC) and 2'-deoxyisoguanosine (isoG) were included at the binding sites of the target, preamplifier, amplifier, and marker-conjugated probes. FA.

B. DNA detection

For DNA detection, harvested cells were fixed with 4% formaldehyde in a phosphate buffer solution (PBS; 0.01 M phosphate buffer, pH 7.5) for 30 minutes on ice, washed twice for two minutes. each and resuspended in PBS. 200 μl aliquots of the fixed cell suspensions were pipetted into double-stained cytospin funnels (Shandon; Pittsburgh, PA) and centrifuged for 6 minutes at 1500 rpm in a cytospin centrifuge (Shandon) using a calculated force equivalent to 300 x g. Cells were dehydrated on slides using a graded series of 70%, 90% and 100% ethanol at room temperature (RT) for 2 minutes each, air-dried at RT for 10 minutes, and stored at -80 ° C for up to 2 weeks. Cells were rehydrated on slides using a graded series of 100%, 90%, and 70% ethanol at RT for

ES 2 287 134 T3 minutes each and washed twice for 2 minutes each in PBS. The slides were incubated at 37 ° C for 1 hour in RNase 40 ng / ml (Sigma) in 2X SSC (1X SSC is 0.15 M NaCl, 0.015 M Na citrate), washed twice for 2 minutes each in PBS and immersed in a preheated jar containing 7.5 to 10 ng / ml Proteinase K (Boehringer Mannheim, Indianapolis, EN) in PBS at 37 ° C for 10 minutes. After washing twice for 2 minutes each in PBS, cells were dehydrated on slides using a graded series of 70%, 90% and 100% ethanol at RT for 2 minutes each and air-dried at RT for 10 minutes. .

Following pretreatment, cells were incubated with a prehybridization solution, hybridized with a set of target oligonucleotide probes (described above), followed by hybridization with a series of oligonucleotide probes for signal amplification. As a prehybridization step, each spot on the slide was incubated with 150 μ. \ Of Hybridization Solution A (3x SSC, 50% formamide, 10% dextran sulfate [MW 500,000], 0.2% casein , poly A 10? ng / ml, denatured salmon sperm DNA 100? ng / ml) at RT for 30 minutes. Slides were transferred to a humidity chamber (Hybaid Omnislide instrument, Phenix Research Products, Hayward, CA), incubated at 92 ° C for 5 minutes, then removed from the chamber and chilled on the bench at RT for 5 minutes. . The prehybridization solution was removed and each spot on the slide was incubated with 100 µl of Hybridization Solution A containing 0.6 pmol of HPV-specific target probes at 40 ° C for 3 hours in the humidity chamber. Following incubation with the target probes, the slides were washed at RT with a decreasing series of SSC buffers containing 0.0025% BRIJ®-35 detergent (SURFACT-AMPS®35, 10%, Pierce, Rockford, IL) such as follows: 3 times for 1-2 minutes with 2X SSC; 3 times for 1-2 minutes with 0.2X SSC; once for 5 minutes with 0.1X SSC; and once for 2 minutes with 2X SSC. Each spot on the slide was incubated with 100 μl Hybridization Solution B (5X SSC, 0.1% to 0.3% sodium dodecyl sulfate [SDS], 10% dextran sulfate, ZnCl₂ 1 mM, MgCl₂ 10 mM) containing 90 fmoles of preamplifier at 55 ° C for 25 minutes in the humidity chamber. The slides were washed twice in 0.1X SSC, 1 mM EDTA for 1 minute and 4 minutes, respectively, and then incubated in 100 μl of Hybridization Solution B containing 90 fmoles of amplifier at 55 ° C for 25 minutes in the humidity chamber. The slides were washed twice in 0.1X SSC, 1 mM EDTA for 1 min and 4 min, respectively, and then incubated in 100 μl Hybridization Solution B containing 90 fmoles of FA-conjugated marker probe at 55 ° C for 15 minutes in the humidity chamber. After washing in D (100 mM tris- (hydroxymethyl) aminomethane, pH 8.0, 0.1% BRIJ®-35, ZnCl₂ 1 mM, MgCl₂ 10 mM) at RT for 5 minutes, 50 µl of buffered FA substrate (Fast Red, # K597, DAKO Corporation, Carpinteria, CA) was added and the slides were incubated at RT for 10 minutes according to the manufacturer's instructions. The slides were rinsed with water three times for 3 minutes each and then counterstained for 40 seconds with Gills 1 hematoxylin (American Histology Reagent Company, Inc., Modesto, CA) or 0.0001% bisbenzimide (No. B2883, Sigma). Slides were mounted with ULTRAMOUNT^® (DAKO Corporation), PERMOUNT^® (Fisher Scientific) or 75% glycerol and stored at RT. The slides were visualized using a Nikon E800 fluorescence microscope with a triple band pass filter or FITC (Nikon, Foster City, CA) or a 60X bright field objective, and the images were captured using a color cooled CCD camera from 3 Optronics chips (Optronics Engineering, Goleta, CA). Fluorescent or chromogenic images were captured using Image Probe software (Media Cybernetics, Silver Springs, MA). Figure 1 is a schematic of the assay format for Example 1.

C. Results

The results of this study demonstrate that after hybridization with the HPV-16 target probes, a positive signal detection was observed in CaSki cells (Figure 2A) and SiHa cells (Figure 2B). As expected, a multitude of signals were detected in the CaSki cells, as they contained approximately 400-600 copies of HPV-16. Only one or two signals were detected in the SiHa cells, also as expected, as they only contained 1-2 copies of HPV-16 DNA. Finally, no signal was detected with HPV-16 probes in cells lacking HPV16, ie, HeLa (Figure 2C) and C33a (Figure 2D) cells. These results demonstrate both the quantitative ability and the specificity of the present procedure.

After hybridization with HPV-18 target probes, a positive signal detection was observed in HeLa cells (Figure 2G), which contained 10-50 copies of HPV-18 DNA. However, no signal was detected with the HPV-18 probes in cells lacking HPV-18, including CaSki (Figure 2E), SiHa (Figure 2F), and C33a (Figure 2H) cells. No signal was detected with the HPV-16 and HPV-18 target probes in the HPV-negative HT3 cell line and the ME180 cell line, which harbor an HPV-39-like DNA sequence (not shown).

Several additional controls were run to demonstrate that the positive signals observed in these experiments were specific for HPV DNA targets. No positive signal was observed when nonspecific target probes were used, nor when HPV-16 or HPV-18 target probes, amplifier, or FA-conjugated marker were omitted from the rDNA HIS procedure. Omission of proteinase K digestion or DNA denaturation steps, or DNase treatment of cells also resulted in signal loss. As an additional control for DNase digestion experiments, the RNase treatment step was omitted to confirm that this loss of signal was not due to DNase degradation of the oligonucleotide probes. Under these conditions, a positive signal was again detected, indicating that the DNA oligonucleotide probes were not degraded and therefore could bind to the HPV RNA targets.

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Example 2

Assessment of specificity in cells

Mixed cell populations were used to further assess the specificity of the present method for the detection of HPV DNA. Mixed cell samples were comprised of unlabeled CaSki cells (containing 400-600 copies of HPV-16) and labeled HeLa cells (containing 10-50 copies of HPV18 DNA). HeLa cells were labeled with the fluorescent cell tracer CFDA SE (Carboxyfluorescein Diacetate, Succinimidyl Ester, Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Both cell types were assayed according to the procedure of Example 1.

As shown in Figures 3A and 3C, hybridization with the HPV-16 target probes produced a positive signal detection only in HPV-16 infected CaSki cells (arrow) and not in HeLa cells (arrow). Also, as shown in Figures 3B and 3D, hybridization with the HPV-18 target probes produced a positive signal detection only in HeLa cells infected with HPV-18 (arrowhead) and not in CaSKi cells (tip arrow). As expected, a higher signal intensity (i.e., greater number and size of spots) was observed for the detection of HPV-16 DNA in CaSki cells compared to the detection of HPV-18 DNA in HeLa cells. . This difference in signal intensity is a reflection of the higher number of HPV DNA copies present in CaSki cells (400-600 copies of HPV-16 DNA / cell) compared to HeLa cells (10-50 copies of HPV-18 DNA / cell).

These results demonstrate that in a mixed population of cells, the present method can distinguish between cells infected with HPV-16 DNA and cells infected with HPV-18 DNA. Furthermore, there is no signal transfer from one cell type to the other since positive signals are maintained only within the appropriate cell types. Furthermore, even low abundance targets can be easily detected with the present method without the background and cross-reactivity problems that could be introduced by the presence of another HPV genotype.

Example 3

Detection of HPV RNA and localization of RNA / DNA within a cell

For RNA detection, cells grown on chamber slides (# 12-565-18, Fisher Scientific, Pittsburgh, PA) were fixed with 4% formaldehyde in PBS for 30 minutes at RT, treated with 10 pg / ml proteinase K in PBS for 10 minutes at RT, and then washed twice for 5 minutes in PBS. Samples were incubated at 40 ° C for 3 hours with 1 pmol of HPV-specific target probes in target probe buffer (6X SSC, 25% formamide, 0.2% BRIJ®-35, 0.2% casein %), and were then washed following the same decreasing series of SSC buffers as described in Example 1. Similarly, the preamplifier, amplifier and FA-conjugated probe hybridization and wash conditions were the same as those described in example 1.

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

FA substrate (Fast Red, DAKO Corporation) was added and the slides were incubated at RT for 4 minutes. After stopping the reaction by washing in PBS, the samples were post-fixed in 4% formaldehyde in PBS for 5 minutes at RT and then contrast stained for 40 seconds with either hematoxylin or bisbenzimide. The slides were viewed and imaged and printed as described in Example 1.

Hybridization of HeLa cells (prepared for RNA detection) with HPV-18 target probes resulted in the detection of HPV-18 mRNA, primarily in the cytoplasm (arrowhead, Figure 4A). However, no signal was observed in these same cells incubated with HPV-16 target probes (Figure 4B).

In contrast, hybridization of HeLa cells (primed for DNA detection) with HPV18 target probes resulted in detection of HPV-18 DNA in HeLa cell nuclei (arrow, Figure 4C). However, no signal was observed in these same cells incubated with HPV-16 target probes (Figure 4D).

These results show that HPV mRNA and HPV DNA are located in different compartments within cells. In particular, viral mRNA is predominantly located in the cytoplasm, while viral DNA is limited to the nucleus. These results also show that positive signals are maintained within the compartment of the cell in which the target nucleic acid is located. In other words, the target and the signal are located together.

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The present method provides a precise subcellular localization, producing positive signals maintained within the subcellular compartments in which the target nucleic acid sequences are located.

Example 4

Detection of HPV-16 by rDNA HIS in CINII samples

A protocol similar to that of Example 1 was used for in situ hybridization in tissues, as follows. Briefly, formalin-fixed paraffin sections of cervix tissue were dewaxed and rehydrated using standard xylene histology and an alcohol graded series. For DNA detection, tissue was digested with RNase at 100 pg / ml RNase in RNase buffer (0.5M NaCl, 10mM Tris pH 9.0, 1mM EDTA) at 37 ° C for 1 hour, followed by proteinase K digestion (12 pg / ml in proteinase K buffer or PBS) at 37 ° C for 10 minutes. Proteinase K was inactivated by post fixation in 4% paraformaldehyde in PBS at 4 ° C for 10 minutes. After several washes in PBS, the tissue was acetylated in 1 M TEA as described in Angerer et al. (1987) In situ Hybridization with RNA Probes-An Annotated Recipe. In Valentine KL et al., Eds., In situ Hybridization-Applications to Neurobiology, Oxford, Oxford University Press: 71-96. Sections were dehydrated in an ethanol series prior to denaturation for 5 minutes at 92 ° C in DB (80% formamide, 2X SSC) over Hybaid (Phenix), followed by immersion in cold 70% ethanol and dehydration. After a 30 minute incubation in a prehybridization buffer, target oligonucleotide probes in fresh prehybridization buffer were added at a concentration of 10 fmol / µl and covered with the coverslip. Hybridization was carried out in a humidified chamber on a Fischer slide warmer at 37 ° C for 1-3 hour (s), with the same washes and signal amplification previously described in Example 1. After hybridization, the sections were washed in a graded series of SSC to 0.1 X SSC for a total of 15 minutes of wash time. Hybridization in 1 fmol / µl of preamplifier in Hybridization Solution B for 25 minutes at 55 ° C was followed by 3 washes in 0.1X SSC for 5 minutes of total wash time. Hybridization in 1 fmol / µl of Amplifier in Hybridization Solution B for 25 minutes at 55 ° C was followed by several washes in 0.1 X SSC. Hybridization in 1 fmol / µl of marker probe in Hybridization Solution B at 55 ° C for 15 minutes was followed by washes in 0.1X SSC and incubation in Wash D. Fast Red was prepared immediately prior to application to cells. sections, and color development was stopped between 4 and 10 minutes. The nuclei were stained with hematoxylin, and the slides were covered with coverslips for microscopy. Endogenous alkaline phosphatase was inactivated with levamisole, 1 pM to 5 pM.

In situ hybridization detected HPV-16 in CIN II (cervical intraepithelial neoplasia) of cervical tissue that has HPV-associated cytopathic changes (sample No. 00B01-627, Clinomics BioSciences, Inc., Pittsfield MA). Cell nuclei in each of the sections were stained with hematoxylin. Figure 5A represents the distribution of HPV-16 gene expression (stained areas) within the squamous epithelium of the ectocervix. Further amplification of the boxed region in Figure 5A shows the presence of HPV-16 mRNA in the cytosol of some squamous epithelial cells (Figure 5B). Figure 5C shows that HPV-16 DNA was detected in the nucleus of squamous cells in a tissue section adjacent to the region inset in Figure 5A. However, HPV-18 DNA was not detected in an adjacent section (Figure 5D). As expected, GAPDH mRNA was detected throughout the squamous epithelium near the transformation zone (Figure E). Figures 5A and 5E have a 200x magnification while Figures 5B, 5C and 5D have a 600x magnification.

Example 5

HPV DNA rDNA HIS with preservation of histopathology

In situ hybridization was carried out using the procedure similar to that of Example 4 to detect HPV-16 DNA in regions of cytopathic changes characteristic of CIN II lesions in cervical tissue that have HPV-associated cytopathic changes (sample No. 00B01 -624, Clinomics BioSciences, Inc., Pittsfield MA), as follows. Cell nuclei were stained in sections with hematoxylin. Large arrowheads indicate contiguous basal cells along the basement membrane separating the squamous epithelium from the underlying stroma. In an apparently normal squamous maturation region, HPV-16 DNA was not detected, the basal cells were bounded near the basement membrane, and the apical part of the epithelium was comprised primarily of differentiated squamous cells with a characteristically large cytosol (Figure 6A). ). A nearby region of the same tissue section showed abnormal squamous maturation characteristic of moderate cervical dysplasia (CIN II) (Figure 6B). This diphasic region showed an overgrowth of basal cells, mixtures of basal cells and differentiated squamous cells in the apical epithelial region, and the presence of HPV-16 DNA (stained area) (Figure 6B). A higher magnification showed the presence of HPV-16 DNA in basal cells, as indicated by smaller arrows (Figure 6C). Figures 6A and 6B have a magnification of 400x and Figure 6C has a magnification of 600x.

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Example 6

Assessment of tissue specificity

In situ hybridization using genotype-specific HPV probes and the procedure of Example 4 was used to detect HPV DNA in CINII tissue sections and in normal cervix sections. Cell nuclei in sections were contrast stained with hematoxylin. The stained areas indicated the presence of the target nucleic acid, viral DNA (Figures 7A, 7B, 7C, 7D, 7E and 7F) or endogenous mRNA (Figure 7G). HPV16 DNA was detected in the squamous ectocervical epithelium of CIN II tissue No. 00B01-624 (Figure 7A), but HPV-18 was not detected (Figure 7B). HPV-18 DNA was detected in CIN II tissue that had HPV-associated cytopathic changes (sample # 00B01-625, Clinomics BioSciences, Inc., Pittsfield, MA) as shown in Figure 7D, but HPV was not detected -16 (Figure 7C). As expected, HPV DNA was not detected in normal cervical tissue (sample # H-1188-88, Clinomics BioSciences, Inc., Pittsfield MA) when tested with HPV-18 probes (Figure 7E) or HPV-16 (Figure 7F). As expected, endogenous GAPDH mRNA was detected in normal cervical tissue (Figure 7G).