WO1995019449A1 - Populations of non-adjacent hybridization probes - Google Patents

Abstract

Probe populations in which probes, labelled with reporter moieties at each of their ends, are designed to hybridize at spaced intervals along a target strand. Additionally, inventions are processes of using the populations.

Description

POPULATIONS OF NON-ADJACENT HYBRIDIZATION PROBES

FIELD OF THE INVENTION

The field of the invention is the detection of nucleic acids by using hybridization procedures.

BACKGROUND The detection of nucleic acids by hybridization procedures is a valuable medical diagnostic procedure. It can, for example, be used to delect viruses, classify microorganisms, and detect genetic defects. In this important procedure, the nucleic acid moiety of a probe is frequently linked lo a delectable reporter moiety so that, after the probe as hybridized lo its target, it can be delected. In some instances, relatively small probes are used, each probe designed to hybridize to a particular target region in the target molecule. In such a case, in order lo double Ihc signal per probe, it can be desirable lo link a reporter moiety, such as a fluorescent moiety, al both the 3' and 5' end of each probe. Nevertheless, if the 3' end of one target region is in very close proximity lo the 5' end of a neighboring target region, the reporter moieties on probes intended to hybridize to neighboring target regions could slcarically interfere with each other, preventing at least one of them from hybridizing. Similarly if two fluorescent moieties al the ends of probes on neighboring target regions are in loo close proximity, there could be a quenching effect as lo fluorescence.

BRIEF SUMMARY OF THE INVENTION

The inventions are probe populations in which probes, labelled with reporter moieties at each of their ends, are designed to hybridize at spaced intervals along a target strand. Related inventions are processes of using the populations.

BRIEF DESCRIPTION OF DRAWING Fig. 1. Map of portion of the human papilloma virus 16 genome. Each row is in the 5' to 3' direction, from lcfl to right. Also shown arc the sequences of Ihe oligonucleotide probes used to create probe populations.

DETAILED DESCRIPTION

DEFINITIONS

"Aminolink 2" is 6-(trifluoroacelylamino)hcxyl-(2-cyanoethyl)-(N,N diisopropyl)- phosphoramidite and is sold by Applied Biosystems, Inc., Foster City, CA, U.S.A. It is used to create a 6-carbon linker moiety that is seven atoms long. "3'-Aminomodifier C7 CPG" sold by Glen Research, Slerling, VA, U.S.A., is (1- dimethoxytrityloxy-3-fluorenylmcthoxycarbonylamino-hexan-2-melhylsuccinoyl)-Iong chain alkyl amino moiety linked to controlled pore glass. Il is used to make a 6-carbon linker moiety that is seven atoms long.

"Aminomodificr II" sold by Clontcch Laboratories, Inc., Palo Alto, CA, U.S.A., is N-Fmoc-O^l-DMT-0²-cyanoclhoxydiisopiOpylaminophosphinyl-3-amino-l,2-propanedioI

(See Nelson, Nucleic Acids Research, vol 17, page 7179 (1989)). It is used lo make a 3- carbon linker moiety that is four atoms long.

An "amplification product" is one generated by an amplification process such as PCR or 3SR, designed lo detect an analytc nucleic acid molecule. The term "dye" includes any molecule or molecular moiety that can be dctccled fluorimetrically or spcctropholomelrically, especially though not necessarily in the visible range of wavelengths.

An "end-labelled probe" is one in which a reporter moiety is covalcnlly linked to the nucleotide or nucleoside located at the 3' or 5' end of the oligonucleotide moiety of a probe. Linkage may be direel (as when an atom of the reporter moiety is directly linked lo an atom of the oligonucleotide moiety) or via a linker group, as when an atom of the reporter moiety is directly linked to an atom of a linker moiety and an atom of the oligonucleotide moiety is also directly linked to an atom of thai linker moiety.

"LCR", "LAR", "LAS", refer lo "ligation chain reaction", "ligation amplification reaction", and "ligalion-bascd amplification system" respectively, reactions which rely on a

DNA ligase to join oligonucleotides that bind to a target (K.J. Barringer ct al, Gene 89, 1 17 (1990); D.Y. Wu and R.B. Wallace, Genomics. 4, 560 (1989)).

A "linker moiety" is a moiety thai links two other moieties ("non-linker moieties") together. The "length" of a linker moiety is measured in atoms and is the number obtained by proceeding one atom at a lime along Ihe linker, starting al the alom linked to one non-linker moiety and counting until the atom linked to the other non-linker moiety, provided that the count obtained is the smallest count possible for that linker and those two nonl inker moieties.

A "moiety" is part of a molecule. For example, a probe molecule may consist of a dye moiety covalently linked lo a linker moiety that is covalenlly linked to an oligounculeotide moiety. The phosphate group (PO₄) at the 5' or 3' end of a nucleic acid moiety is considered to be part of the nucleic acid moiety, independent of how il became part of the nucleic acid moiety during the procedure used to synthesize the probe molecule. "PCR" refers to the polymerase chain reaction, an amplification process that uses oligonucleotide primers and a Taq polymerase, (see, for example, PCR protocols: a Guide to Methods and Applications. M. A. Innis et al., Eds., Academic Press, San Diego, California, 1990).

"Qβ replicase" system uses thai RNA bacteriophagc enzyme to effect amplification. (P. M. Lizardi et aj. Biotechnology 6, 1197 ( 1988)).

"TAS" is a transcription-based amplification system that uses oligonucleotide primers, a reverse transcriptasc, and DNA-dependenl RNA polymerase. (D.Y. Kwoh et al. Proc Natl. Acad. Sci. USA, 86, 1 173 (1989)) "3SR" is an amplification system that uses oligonucleotide primers, a reverse transcriplase, DNA-dependenl RNA polymerase, and RNase II (J.C. Gualclli ct a[, Proc. Nail. Λcad. Sci. USA, 87, 1874 (1990).)

A "target molecule" is (he nucleic acid molecule to which a probe is intended to hybridize as a result of that probe's base sequence. A target molecule may be the analyte molecule (the molecule which (he assay is designed lo delect) or a molecule, such as an amplification product, whose presence indicates the presence of an analyte molecule.

ASPECTS OF THE INVENTION In one general aspect, the invention is a process of detecting a nucleic acid target molecule, said process comprising the steps of:

1) incubating, in a solution, a nucleic acid target molecule and a population of probe molecules, wherein the base sequences of Ihe probe molecules will allow them to hybridize to discrete separated target regions along said target molecule, and 2) detecting probe molecules that have hybridized to said target molecule, the separation between said target regions being 5 lo 25 bases on average, wherein for each probe molecule, a first reporter moiety is linked either directly or via a linker moiety indireclly lo the nucleoside al the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked either directly or via a linker moiety indireclly lo the nucleoside at the 3' end of said probe molecule, wherein the sum of the molecular weights of said first reporter moiety and said first linker moiety does not exceed 2000, and wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety does not exceed 2000. It is preferred that the lcnglh of the linker not exceed 10 atoms. It is understood that the hybrid molecule comprising a probe molecule and a target molecule is formed because the nucleic acid moiety of the probe has a base sequence complementary to a base sequence of the target molecule (e.g., the adenine complementary to either uracil or thymine in the other strand, guaninc complementary to cytosinc in the other strand), ll is not necessary, however, that the entire nucleoside sequence of the probe be complementary to Ihe entire nucleoside sequence of the target.

In another aspect, the invention is a process of delecting a nucleic acid target molecule, said process comprising the slcps of: 1) incubating, in a solution, a nucleic acid target molecule and a population of probe molecules, said target molecule comprised of a first strand and a second strand, said second strand complementary in base sequence lo said first strand, said probe molecules comprising base sequences that allow them lo hybridize both to discrete separated target regions on the first strands of said target molecule and discrete separated target regions on the second strand of said target molecule, and

2) detecting probe molecules that have hybridized to a strand of said target molecule, the separation between said target regions on the first strand being 5 to 25 bases on average, the separation between said target regions on the second strand being 5 lo 25 bases on average, each ten-base sequence at the end of a discrete target region on the first strand strand being complementary in base sequence to a ten-base sequence at an end of a discrete target region on the other strand, thereby creating an overlapping pattern of discrete target regions in the target molecule, provided that target sequences at the ends of the pattern may overlap cither one or two target sequences from the other strand, wherein for each probe molecule, a first reporter moiety is linked either directly or via a linker moiety indirectly to the nucleoside al the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked cither directly or via a linker moiety indirectly to the nucleoside at the 3' end of said probe molecule, wherein the sum of the molecular weights of said first reporter moiety and said first linker moiety does not exceed 2000, and wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety does not exceed 2000.

In still another aspect, Ihe invention is a population of probe molecules, wherein the base sequences of the probe molecules will allow them lo hybridize to discrete separated target regions along a target molecule, the separation between Ihe target regions being 5 lo 25 bases on average, wherein for each probe molecule, a first reporter moiety is linked cither directly or via a linker moiety indireclly lo the nucleoside at the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked cither directly or via a linker moiety indirectly to the nucleoside at the 3' end of said probe molecule, wherein the sum of the molecular weights of said first reporter moiety and said first linker moiety does not exceed 2000, and wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety does not exceed 2000, said target molecule having a base sequence identical lo or complementary to a nucleic acid molecule found in a biological entity that is a cell or virus.

PROBES

The nucleic acid moiety of a probe molecule is normally single-stranded and may be DNA, RNA. The DNA or RNA may be composed of Ihe bases adenosine, uridinc, thymidine, guanine, cytosinc, or any natural or artificial chemical derivatives thereof.

Probes are end-labeled with a detectable reporter moiety for use in practicing the invention. Typical reporter moieties are fluorescers (see Clin. Chem., 25_^:353 (1979)) chromophores; luminescers such as chcmiluminescers and bioluminescers (see Clin. Chem., 25:512 (1979)); specifically bindable ligands; and reporter moieties that are radioactive because part of their structure is a radioisotope such as ³H, ³⁵S, ³²P, ^l25I and C. Other reporter moieties arc ones that are enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Patents Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Patent No. 4,134,792). Biotinylated probes (e.g. by Pholobiotin™ labeling of probes) are detected after hybridization using avidin/strcptavidin, fluorescent, enzymatic or colloidal gold conjugates. Nucleic acids may also be labeled with immunodetectable reporter moieties.

Nucleic acid probes can be used against a variety of nucleic acid targets, viral, prokaryotic, and eukaryotic. The target can, for example, be a gene (e.g., oncogene), a control element (e.g., promoter, reprcssor, or enhancer), mRNA, a sccμicnce coding for ribosomal RNA, transfer RNA, or RNase P. The target may be an amplification product, such as the product of PCR, 3SR, TΛS, ligation chain reaction, ligation amplification reaction, ligation-based amplification system, or Qβ replicase system. The target may be viral DNA or RNA. Viral RNA includes RNA that is genetic material, mRNA, and non- genetic material complementary to mRNA. Viral DNA includes genetic material (e.g., in "DNA viruses") and the product of reverse transcriptasc (cDNA) or DNA copies thereof.

The sccμienccs of many viral nucleic acids, such as human immunodeficiency virus and human papilloma virus arc published and/or available through the GcnBank database.

A viral nucleic acid can be part of a virus, in which case the virus may or may not be inside a cell. Alternatively, a viral nucleic acid target may not be part of a virus, but may be inside a cell.

Preferably the reporter moiety is a dye molecule; especially preferred is that the reporter moiety be a fluorescent dye moiety. A fluorescent dye can be detected in a flow cytometer or under a microscope fitted for detection of fluorescence.

Preferred dyes for use as reporter moieties are fluorescent dyes that absorb light in the visible range and emit light in the visible range.

The target nucleic acid molecule can be a purified nucleic acid molecule or one located in a biological entity. A biological entity can be a cell or a virus. The cell or virus may be one that has been treated with a fixative. β

When the reporter moiety is a fluorescent dye, probes bound lo the target arc delected by exposing the target-bound probe to light at a wavelength thai is absorbed by Ihe dye, and delecting the light emitted by ihe dye moiety.

An example of a reporter group that can be detected by a nonfluorcsccnt method is biotin, which can be detected on the basis of its ability lo bind lo a second compound, streptavidin, which in turn can be linked to enzymes such as alkaline phosphalase or horse radish peroxidase that are delectable on the basis of their ability lo react wilh a substrate. Moieties that participate in chcinilumincsccnt reactions ( e.g., 3-aminophthalhydrazidc ("luminol"), 4-methoxy-4-(3-phosphalcphcnyl)-spiiO-(l ,2-dioxctanc-3,2'-adamantanc) disodium salt}, and moieties that react with antibodies arc also among the possibilities for reporter moieties.

In addition to Aminolink 2, 3-Amino-Modifier C7 CPG, and Λminomodificr II, compounds that can be used lo create linkers of various lengths can be synthesized or purchased, for example, from Clontech Laboratories, Inc. {e.g., 3-Amino-Modifier C3 CPG, 5'-Amino-Modifier C3, or 5'-Amino-Modifier 5, or Cn-Aminomodifiers such as N- monomcthoxytrityl O-methoxydiisopropylaminophosphinyl 3-aminopropan(l)ol , and N- monomclhoxytrilyl 3-aminopιopan(l )ol (Sec Connoly, Nucleic Acids Research, vol 15, page 3131 (1987)); generally see Clonctcch's manual "DNA Modification Reagents for Use in Automated DNA Synthesis, A Users Manual (1989), including the references as to methods of synthesis on pages 16-17)}.

For probes used lo delect targets in the cell nucleus (e.g., nuclear DNA) it is preferred that the probe molecules have no more than 40 nucleotides. A probe should normally be at least about 15 bases long for specific hybridization to take place. WHEN THE TARGET MOLECULE IS A PURIFIED NUCLEIC ACID

A purified nucleic acid is considered here to be one that has been extracted from a cell or has been synthesized in vitro in a cell-free system. Many procedures have been published for hybridizing probes to such purified nucleic acids. Generally, if the target is a DNA molecule, its strands are separated by heal or other means before the hybridization step takes place. Such hybridization can take place with the purified target nucleic acid immobilized on a solid support (e.g., nitrocellulose paper for DNA, nylon for RNA) by well-established procedures.

The hybridization conditions vary considerably, depending in part on the level of specificity desired. Some examples arc the Southern Blot procedure ( J. Mol. Biol., 98,

503-517 ( 1975)) for elcctrophorcscd and immobilized DNA, Ihe Northern Blot procedure (Seed, B., in Genetic Engineering: Principles and Methods, Setlow, J.K. and Hollaender, A., eds., 1982; P. S. Thomas, Proc. Nail. Λcad. Sci. USA., 77: 5201 ( 1980)) and the use of stringent conditions with short oligomcr probes, (S. V. Suggs ct al, Proc. Nail. Acad. Sci. USA, 78, 6613-6617 (1981)).

TARGET MOLECULES IN CELLS, TISSUE, AND FLUIDS

The hybridization assay can also be done for target molecules in biological entities. The biological entity can be a cell or a virus and be cither in liquid suspension, on slides or other solid supports, in ti.ssuc culture, or in tissue sections. When the biological entity is a cell, it can come from solid tissue (e.g., nerves, muscle, heart, skin, lungs, kidneys, pancreas, spleen, lymph nodes, tcstcs, cervix, bone marrow, and brain) or cells present in membranes lining various tracts, conduits and cavities (such as the gastrointestinal tract, urinary tract, vas defcrens, uterine cavity, uterine tube, vagina, respiratory tract, nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi and lungs) or cells in an organism's fluids (e.g., urine, stomach fluid, sputum, blood and lymph fluid) or stool. WHEN THE TARGET MOLECULE IS IN A BIOLOGICAL ENTITY

Two useful summaries of possible hybridization conditions arc in PCT International Patent Applications with publication numbers WO 90/02173 and WO 90/02204, and U.S. patent 5,225,326. Iii situ hybridization allows the detection of RNA or DNA sequences within individual cells. With sufficiently large targets, it can detect as few as 1-5 target molecules per cell (PCT Applications WO 90/02173 and WO 90/02204). It also allows for the simultaneous detection of more than one different polynucleolidc sequence in an individual cell. It also allows detection of proteins and polynuclcotides in the same cell. Many different hybridization conditions (solvent composition, temperature, lime) are possible. The ones mentioned below arc only intended to advise the reader of some of the more preferable hybridization conditions. A person skilled in the art will know many other conditions could also be used effectively.

The hybridization step may, for example, be carried out in a solution containing a chaotropic agent such as 50% formamide, a hybrid stabilizing agent such as five times concentrated SSC solution (lx = 0.15 M sodium chloride and 0.015 M sodium citrate), a buffer such as 0.1M sodium phosphate (pll 7.4), about 100 micrograms (ug)/millililer (ml) low molecular weight DNA to diminish non-specific binding, 0.1 % Triton X-100 to facilitate probe entry into the cells and about 10-20 mM vanadyl ribonucleoside complexes.

All percentages for liquids arc on a v/v basis unless otherwise noted. To the hybridization solution is added a probe population designed to hybridize with the target nucleic acids. If the cells are to be ultimately viewed on glass slides (or other solid supports), the cells as either single cell suspensions or as tissue slices are deposited on the slides. The cells are fixed by choosing a fixative which provides the best spatial resolution of the cells and the optimal hybridization efficiency. After fixation, the support bound cells may be dehydrated and stored at room temperature or the hybridization procedure may be carried out immediately.

The hybridization solution containing the probe is added in an amount sufficient to cover the cells. The cells are then incubated at an appropriate temperature. Conditions where preferred temperatures are in the range 5ϋ"-55"C have been disclosed in PCT applications WO 90/02173 and WO 90/02204. However, temperatures ranging from 15°C. to 80°C. may be used.

The hybridization solution may include a chaotropic denaturing agent, a buffer, a pore forming agent, a hybrid stabilizing agent, and the target-specific probe molecule.

The chaotropic denaturing agents (Robinson, D. W. and Grant, M. E. (1966) J. Biol. Chem. 241: 4030; Hamaguchi, K. and Geiduscheck, E. P. (1962) J. Am. Chem. Soc. 84: 1329)) include formamidc, urea, thiocyanalc, guanidine, trichloroacctatc, tetramethylamine, perchloratc, and sodium iodide. Any buffer which maintains pll al least between 7.0 and 8.0 is preferred.

The pore forming agent is for instance, a detergent such as Brij 35, Brij 58, sodium dodccyl sulfate, CHAPS™ Triton X-100. Depending on the location of the target molecule, the porc-fonning agent is chosen lo facilitate probe entry through plasma, or nuclear membranes or cellular compartmental structures. For instance, 0.05% Brij 35 or 0.1 % Triton X-100 will permit probe enlry through the plasma membrane but not the nuclear membrane. Alternatively, sodium desoxycholate will allow probes to traverse the nuclear membrane. Thus, in order to restrict hybridization to the cytoplasmic biopolymcr targets, nuclear membrane pore-forming agents arc avoided. Such selective subccllular localization contributes to the specificity and sensitivity of the assay by eliminating probe hybridization lo complementary nuclear sequences when the target biopolymcr is located in the cytoplasm. Agents other than detergents such as fixatives may serve this function. Purlhcrmorc, a biopolymcr probe may also be selected such that its size is sufficiently small to traverse the plasma membrane of a cell but is too large to pass through the nuclear membrane. Hybrid stabilizing agents such as salts of mono- and di-valcnt cations are included in the hybridization solution lo promote formation of hydrogen bonds between complementary nucleotide sequences of the probe and its target biopolymcr. Preferably sodium chloride at a concentration from .15 M lo 1 M is used. In order to prevent non-specific binding of nucleic acid probes, nucleic acids unrelated lo the target biopolymers are added to the hybridization solution at a concentration of about 100-fold the concentration of the probe. Specimens are removed after each of the above steps and analyzed by observation of cellular morphology as compared to fresh, untreated cells using a phase contrast microscope. The condition determined to maintain the cellular morphology and the spatial resolution of the various subccllular structures as close as possible to the fresh untreated cells is chosen as optimal for each step.

Prior to nucleic acid hybridization, the cells may be reacted with antibodies in phosphate buffered saline. After hybridization one may analyze the cells for both bound antibodies and bound hybridization probes.

MOUNTING BIOLOGICAL ENTITIESπTSSUES

Many types of solid supports may be utilized to practice the invention. Supports which may be utilized include, but are not limited to, glass, Scotch tape (3M), nylon, Gene Screen Plus (New England Nuclear) and nitrocellulose. Most preferably glass microscope slides are used. The use of these supports and the procedures for depositing specimens thereon will be obvious to those of skill in the art. The choice of support material will depend upon the procedure for visualization of cells and the quantilalion procedure used. Some filter materials are not uniformly thick and, thus, shrinking and swelling during jn situ hybridization procedures is not uniform. In addition, some supports which aulofluoresce will interfere with the determination of low level fluorescence. Glass microscope slides are most preferable as a solid support since they have high signal-to-noisc ratios and can be treated lo better retain tissue.

FIXATION OF BIOLOGICAL ENTITIES/TISSUES

A fixative may be a precipitating agent or a cross-linking agent used alone or in combination, and may be aqueous or non-aqueous. The fixative may, for example, be selected from the group consisting of formaldehyde solutions, alcohols, salt solutions, mercuric chloride sodium chloride, sodium sulfate, potassium dichromate, potassium phosphate, ammonium bromide, calcium chloride, sodium acetate, lithium chloride, cesium acetate, calcium or magnesium acetate, potassium nitrate, potassium dichromate, sodium chromatc, potassium iodide, sodium iodate, sodium thiosulfate, picric acid, acetic acid, paraformaldehyde, sodium hydroxide, acetones, chloroform, glycerin, thymol, etc. Preferably, the fixative will comprise an agent which fixes the cellular constituents through a precipitating action and has Ihe following characteristics: the effect is reversible, the cellular (or viral) morphology is maintained, the antigenicily of desired cellular constituents is maintained, the nucleic acids are retained in the appropriate location in the cell, the nucleic acids are not modified in such a way thai they become unable to form double or triple stranded hybrids, and cellular constituents are not affected in such a way so as to inhibit the process of nucleic acid hybridization lo all resident target sequences. Choice of fixatives and fixation procedures can affect cellular constituents and cellular morphology; such effects can be tissue specific. Preferably, fixatives for use in the invention include ethanol, ethanol-acetic acid, methanol, and methanol-acetone. Preferred fixatives afford high hybridization efficiency with good preservation of cellular morphology.

Fixatives for practicing Ihe present invention include 95% cthanol/5% acetic acid for IIL-60 and normal bone marrow cells, 75% ethanol/20% acetic acid for K562 and normal peripheral blood cells, 50% melhanol/50% acetone for fibroblast cells and normal bone marrow cells, and 10% formaldehyde/90% melhanol for cardiac muscle tissue. Ethano mcthanol (3: 1 , v/v) can be used in all types of cells. These fixatives provide good preservation of cellular morphology and preservation and accessibility of antigens, and high hybridization efficiency.

Simultaneously, the fixative may contain a compound which fixes the cellular components by cross-linking these materials together, for example, glutaraldchydc or formaldehyde. While this cross-linking agent must meet all of the requirements above for the precipitating agent, it is generally more "sticky" and causes the cells and membrane components to be secured or sealed, thus, maintaining the characteristics described above.

The cross linking agents when used are preferably less than 10% (v/v). Cross-linking agents, while preserving ultrastructure, often reduce hybridization efficiency; they form networks trapping nucleic acids and antigens and rendering them inaccessible to probes and antibodies. Some also covalcnlly modify nucleic acids preventing later hybrid formation. H

STORAGE OF BIOLOGICAL ENTITIES/TISSUES

After fixation, microscope slides containing cells may be stored air dried at room temperature for up lo three weeks, in cold (4"C) 70% ethanol in water for 6- 12 months, or in paraplast for up to two years. If specimens arc handled under RNase-free conditions, they can be dehydrated in graded alcohols and stored for at least 5 months at room temperature.

Reagents can be purchased from any of a variety of sources including Aldrich Chemical Co., Milwaukee, Wisconsin, Sigma Chemical Co., Si. Louis, Missouri, Molecular Probes, Inc., Eugene, Oregon, Clontcch, Palo Alto, California, Kodak, Rochester, NY, and Spectrum Chemical Manufacturing Corp., Gardcnea, California.

DETECTION OF ONCOGENES IN PERIPHERAL BLOOD CELLS AND BONE MARROW CELLS

In a typical procedure, 10 ml. of human peripheral blood or 2 ml. of human bone marrow cells are incubated at 37 °C. in a 1.2% (215 mOs) ammonium oxalate solution to lyse the red blood cells. The white blood cells are centrifuged at 3,000 rpm for 10 minutes in a clinical centrifuge. The cell pellet is subsequently washed with 10 l. PBS and the pellet is resuspended in PBS. Cells are deposited by cytoccnlrifugation onto precleaned glass slides and air dried for 5 min. The cells arc then fixed in 75% ethanol/ 20% acetic acid for 20 min. at room temperature. Hybridization procedures using oncogene-specific probes arc then followed.

HYBRIDIZATION IN SOLID TISSUE

In a typical procedure, lour micron thick frozen sections of human breast tissue obtained from surgically removed biopsy samples are mounted on precleaned glass slides and fixed with 50% methanol/50% acetone for 20 in. al room temperature. Hybridization then proceeds using procedures described elsewhere in this document. 16

ONE-STEP IN SITU HYBRIDIZATION ASSAY

Briefly, cells, either as single cell suspensions or as tissue slices may be deposited on solid supports such as glass slides. Alternatively, cells arc placed inlo a single cell suspension of about 10^s- 10° cells per ml. The cells are fixed by choosing a fixative which provides the best spatial resolution of the cells and the optimal hybridization efficiency.

The hybridization is then carried out in the same solution which effects fixation. This solution contains both a fixative and a chaotropic agent such as formamide. Also included in this solution is a hybrid stabilizing agent such as concentrated lithium chloride or ammonium acetate solution, a buffer, low molecular weight DNA and/or ribosomal RNA (sized to 50 bases) lo diminish non-specific binding, and a pore forming agent lo facilitate probe entry into the cells. Nucleasc inhibitors such as vanadyl ribonuclcoside complexes may also be included. To the hybridization solution is added a probe (or probes), lo hybridize with a target polynucleotide.

The one-step procedure is a means of carrying out the fixation, prehybridizalion, hybridization and detection steps normally associated witli in situ hybridization procedures all in one step. By modifying (lie components of this "one-step" solution, a convenient tcmperalure may be used to carry out the hybridization reaction. Furthermore, this provides a hybridization assay which can be accomplished with viable or non-viable cells in solution. In either case, the assay is rapid and sensitive. Regardless of whether the cell specimen is in suspension or on solid supports, the one-step hybridization procedure is carried out utilizing a single hybridization solution which also fixes the cells. This fixation is accomplished in the same solution and along with the hybridization reaction. The fixative may be selected from the group consisting of any precipitating agent or cross-linking agent used alone or in combination, and may be aqueous or non-aqueous.

Tissue samples are broken apart by physical, chemical or enzymatic means into single cell suspension. Cells arc placed into a PBS solution (maintained to cellular osmolality with bovine serum albumin (BSA) al a concentration of 10^s lo 10^ft cells per ml. Cells in suspension may be fixed and processed at a later lime, fixed and processed immediately, or not fixed and processed in the in situ hybridization system of the present invention.

A single solution is added lo the cells/tissues (hereafter referred lo as the specimen). This solution contains the following: a mild fixative, a chaotropc, a nucleic acid probe (RNA or DNA probe which is prelabcled) and/or antibody probe, salts, detergents, buffers, and blocking agents. The incubation in this solution can be carried out al 55"C for 20 minutes as well as other conditions such as those in the Example below.

The fixative is preferably one that has been found lo be optimal for the particular cell type being assayed (eg., there is one optimal fixative for bone marrow and peripheral blood even though this "tissue." contains numerous distinct cell types). The fixative is usually a combination of precipitating fixatives (such as alcohols) and cross-linking fixatives (such as aldehydes), with the concentration of the cross-linking fixatives kept very low (less than 10%). Frequently, the solution contains 10-40% ethanol, and 5% formalin. The concentration and type of precipitating agent and crosslinking agent may be varied depending upon the probe and the stringency requirements of the probe, as well as the desired temperature of hybridization. Typical useful precipitating and cross-linking agents are specified in PCT applications WO 90/02173 and WO 90/02204.

The hybridization cocktail contains a denaturing agent, usually formainide at about 30% (v/v), but other chaotropic agents such as Nal, urea, etc. may also be used. Furthermore, several precipitating and/or cross-linking fixatives also have mild denaturing properties; these properties can be used in conjunction with the primary dcnalurant in cither an additive or synergistic fashion. The hybridization cocktail may be constructed to preferentially allow only the formation of RNA-RNΛ or RNA-DNA hybrids. This is accomplished by adjusting the concentration of the denaturing agents along with the concentration of sails (primarily monovalcnt cations of the Group I series of metals along with the ammonium ion) and along with the temperature of hybridization which is used. This allows for the selective hybridization of probe to either cellular RNA or DNA or both RNA and DNA simultaneously with distinct probes. This further allows the probes to be supplied in a premixed solution which presents the optimal conditions for generating a signal and minimizing noise while simultaneously optimally "fixing" the morphology of the cells/tissues.

ADDITIONAL USEFUL REAGENTS AND SOLUTIONS Useful reagents and solutions for performing hybridization include 0.0025% Evans

Blue and/or 10% sodium dodecyl sulfate in the solution analyzed cytofluorimetrically; 5% (v/v) vitamin E in the hybridization cocktail used when the target is a biological entity; about 8% DMSO (v/v) with about 5% or 10% squalane and about 5% or 10% pyrrolidinone in the hybridization cocktail when the target is in a biological entity; 5 ul of 1 M (1 molar) dithiothreitol and 5 ul of Proteinase K (1 mg/ml) solution are added to 100 ul of cocktail and the hybridization reaction is run, for example, at 42°C for 5 min, then at 95°C for 5 min, and then at 42°C for 2 min, when the target molecule is in a biological entity; and/or about 0.05% or 0.10% aurintricarboxylic acid (or 1-10 mg/ml basic fuchsin) in the hybridization cocktail when the target molecule is in a biological entity and a fluorescent reporter moiety, especially fluorescein or a rhodamine derivative is used.

When short probes are used, probes against both strands of a double-stranded target can be used without probes hybridizing to each other, provided that a probe that hybridizes to one strand is complementary in base sequence to no more than 15 bases of a probe that hybridizes to the other strand, a situation that can be achieved by having the target base sequences along one strand out-of-phase as to the target sequences along the other strand.

KITS

The present invention may be provided in the form of a kit. Therefore, another invention is a kit for detecting a target nucleic acid molecule in a biological entity, said kit comprising a probe population described herein and one or more reagents (selected from the group, a fixative and a chaotropic agent) for use in a solution for reacting said probe population with said biological entity so that a hybrid molecule can form between a molecule of the probe population and a nucleic acid target molecule in the biological entity. For example, a kit could include a solution containing a fixation/hybridization cocktail and one or more probe populations of this invention. This solution could, for example, contain 15-40% ethanol, 25-40% formamide, 0-10% formaldehyde, 0.1-1.5 M LiCl , 0.05-0.5 M Tris-acetale (pll 7-8), 0.05%-0.15% Triton X-100, 20 ug/ml-200 ug/ml of a non-specific nucleic acid which docs not react with Ihe probe(s), and 0.1 ug/ml to 10 ug/ml of single stranded probes directly labeled with a reporter molecule. More specifically, for example, this solution could contain 30% ethanol, 30% formamide, 5% formaldehyde, 0.8M LiCl , 0. I M Tris-acclalc (pl l 7.4), 0.1 % Trilon X- 100, 50 ug/ml of the non-specific nucleic acid, and 2.5 ug/ml of each single stranded probes directly labeled with a fluorescent reporter molecule. Additionally, there would preferably be means and instructions for performing the said in situ hybridization reaction of the present invention.

Alternatively, the kil could include concentrated stock solution(s) lo be diluted sufficiently to form the solutions needed for hybridization. Additionally, it could include any mechanical components which may be necessary or useful to practice the present invention such as a solid support (e.g. a microscope slide), an apparatus lo affix cells to said support, or a device to assist with any incubations or washings of the specimens, and/or a photographic film or emulsion with which lo record results of assays carried out with the present invention. Normally, the kil would include instructions on how to carry out the hybridization.

FLUORESCENT MEASUREMENTS

Fluorescent measurements can be made using a fluorescent microscope such as an Olympus BH10 microscope wilh fluorescent capabilities. Fluorescent measurements can also be made on a flow cytometer, such as a FACSTAR™ made by Becton Dickinson. EXAMPLES

Example 1

Synthesis of an oligonucleotide with Aminomodifier II at its 5' end and no dye al its 3' end

To create oligonucleotidcs wilh a dye linkers al the 5' end only, a DNA synthesizer is used. DNA synthesizers and literature describing the required phosphoroamidilc chemistry can be obtained from various sources including Applied Biosyslcms, Inc.

("ABI"), Foster City, CA, Glen Research, Sterling, VA, National Bioscicnces, Inc., Plymouth, MN, Cruachem, Dulles, VA, Biogenex, San Ramon, CA, Milligen, Watertown, MA, and Pharmacia, Piscataway, NJ (Sec, for example, ABI DNΛ/RNA Synthesizers User's Manual, Models 392,394, Doc. Rev. A., Applied Biosyslcms, August, 1992; ABI User Bulletin, Model 370, Issues 11, Jan. JO, 1989; Nature 32J. 674 (1986)). The automated synthesizer is used in Ihe same way that it is used to normally (no dye linkers) create an oligonucleotide by standard phosphoramidile chemistry except thai a linker is added instead of a nucleoside in the last synthesis step. More specifically the steps are as follows: 1) A DNA Synthesizer is equipped wilh a support made, for example, of controlled-pore glass (CPG) or polystyrene; the CPG is attached via a linker to a nucleoside whose 5' hydroxyl is blocked with dimcthoxylrilyl (DMT). That nucleoside will be the 3' nucleoside of the complete oligonucleotide.

2) The 3 'nucleoside is delritylated so as create a 5' hydroxyl group and that group is reacted wilh a DMT-protectcd phosphoramidile nucleoside.

3) Subsequent nucleosides are added by repeating step (2).

4) The oligonucleotide is then detrytilated and ils resulting 5' hydroxyl group is reacted with the phosphoramidile compound, Aminomodifier II (Clonetcch Laboratories, Inc.), instead of another nucleoside. 5) Continuing wilh the DNA synthesizer as in a normal oligonucleotide cycle, all Ihe protecting groups (e.g., the li fluorocarbonyl group on the Aminomodifier 11, and the groups protecting the phosphate groups) arc removed and the oligonucleotide is cleaved from the solid support linker. The cleavage creates an oligonucleotide wilh a a 3' hydroxyl group and a 5' primary amine linker attached lo an oxygen atom of the 5' phosphate group of the 5' nucleoside. (Clonetech Laboratories, Inc., Manual " DNA Modification Reagents for Use in Aulomomated DNA Synthesis", Pages 2-3, 1989. The primary amine group of Ihe aminoethyl linker is available for reaction with a fluorescent dye.

Example 2

Synthesis of an oligonucleotide wilh Aminolink 2 at its 5' end and a 3'-Amino- Modifier C7 CPG linker at its 3' end

To create oligonuclcotidcs wilh dye linkers at the 5' and 3' ends, a DNA synthesizer is used. The automated synthesizer is used in the same way is used to normally (no dye linkers) create an oligonucleotide by standard phosphoramidile chemistry, except that special linkers link the oligonucleotide lo the column and a linker is added instead of an nucleoside as the last base. More specifically the slcps are as follows:

1) A DNA Synthesizer is equipped wilh ( l-dimelhoxylrityloxy-3- fluorenylmethoxycarbonylamino-propan-2-succinoyl)-long chain alkylamino-CPG ("3'-

Amino-Modifier C7 CPG") sold by Glen Research (Sterling, VA) is used.

2) The 3'-Amino Modifier C7 CPG is detrilylated so as to create an hydroxyl group and that group is reacted with a phosphoramidite nucleoside.

3) Subsequent nucleosides are added by standard DNA Synthesizer phosphoramidite chemistry. 4) After the oligonucleotide is synthesized, and then the phosphoramidite compound Aminolink 2 reacts with the oligonuclcotide's 5' hydroxyl group in the same manner as a protected phosphoramidile nucleoside would react wilh that hydroxyl during normal oligonucleotide synthesis by the DNA synthesizer. The phosphate group created by that reaction is protected by a methyl group.

5) Continuing wilh the DNA synthesizer as in a normal oligonucleotide synthesis cycle, all the protecting groups (including Ihc trifluorocarbonyl group on the Aminolink 2 and the groups protecting the phosphate groups) arc removed and the oligonucleotide is cleaved from Ihc CPG column. The cleavage crcales an aminohexy linker attached to an oxygen atom of the 3' phosphate group of the oligonucleotide and an aminohexyl linker attached to an oxygen atom of Ihc 5' phosphate group of Ihc 5' nucleoside. The primary amine groups of the linkers are available for reaction with a fluorescent dye.

Example 3

Synthesis of an oligonucleotide wilh Aminomodifier II at ils 5' and a 3'-Amino- Modifier C7 CPG linker al its 3' end

The procedure of Example 2 is followed except that Aminomodifier II (from Clonetech Laboratories, Inc.) is used instead of Aminolink 2. Groups eliminated by the deprotection step include the fluorenylmelhoxy carbomyl ("Fmoc") group contributed by the 3'-Amino-modifier C7 CPG. Example 4

Addition of dye molecules to the linker primer amine groups at the ends of oligonucleotides

Labeling Procedure for oligonucleotide with free amines on the 3' and 5' ends.

1. Resuspend 25 mg of TAMRA in 400 μl of dry DMSO. Vortex to mix. (TAMRA is 5- (and -6) -carboxytetramethylrhodamine succinimidyl ester; sold by Molecular Probes, Cat No. 1171)

2. Resuspend 500 μg of dried oligonucleotide in 50 μl of water. Vortex to mix.

3. Add 50 μl of carbonate buffer (pH 9.0) and 32 μl of the TAMRA/DMSO solution to the oligonucleotide. Mix by vortexing.

4. Microfuge at 12,000 xg for 15 seconds.

5. React for 3 hours at room temperature (about 23 °C).

6. Repeat steps 1-4.

7. React for 8 to 16 hours at room temperature.

8. Precipitate the labeled oligonucleotide by adding 0.1 x total volume of 3M sodium acetate (pH 5.2) and 2.5 x total volume of cold ethanol (4° C). Vortex and chill at - 20° C for 30 minutes. After 30 min., spin at 12,000 xg for 20 minutes. Decant ethanol and save pellet. Dissolve pellet in 50 ul of water.

9. Repeat step 8. 10. Resuspend the pellet in 50 μl water and pass through a Sephadcx G-50 exclusion column. Collect the first colored band (DNA) and dry using a vacuum concentrator.

1 1. Using Reversed phase UPLC, purify the labeled DNA lo remove the unconjugated, truncated and branched-chaincd material.

12. Dry the fractions down, Ihcn ethanol precipitate each of ihc fractions as in step 8.

13. Run the fraction on a 20% polyacrylamidc gel and select only fractions containing full length oligomcrs.

For an oligonucleotide that just has a 5' amine modification, do the exact same procedure except skip step 6. The primary amine of a linker displaces the N-hydroxy succinimidc group located at the TAMRA carbonyl group carbon atom, thereby creating an amide linkage.

Example 5 Demonstration that Fluorescent Signal Intensity is Doubled when Both the 5'-end and 3'-cnd Nucleosides Are Labelled and a 5-Base Interval Exists Between Neighboring Probes on the Target

The cells used were CASKI cells (American Type Culture Collection # CRL1550) containing 400-500 copies of the IIPV 16 genome inlcgraled into Ihc cellular genome and, as a negative control, C-33A cells (ATCC #HTB31). Cells were grown to confluence in 5% C0 , then rinsed in IX PBS. To the cells were added 0.25% trypsin in 0.02 M EDTΛ. They were incubated at 37°C for 5 min, then gently tapped to dislodge cells. They were then washed in media and then spun by cytospin for about 7 min at 700 rpm onto clean glass slides and left to air dry. To the dried cells was added 20 ul of cthanohmelhanol (3: 1 ). They were then allowed to dry.

Hybridization was done by incubating the cells on slides in 20 ul of a hybridization cocktail al 42 °C for 5 min, Ihen 90 "C for 5 min, then 42 "C for 20 min after which the cells were washed once with wash solution A and five times wilh wash solution 13 before being mounted in mounting solution. The hybridization solution was made according lo the following formula:

500X Ficoll/PVP (0.3ml) 4M Guanidine thiocyanate (1.0 ml) 0.5 M EDTA buffer (pll 8.0) 30 X SSC (1.66 ml) 1 M sodium phosphate buffer (0.5 ml)

5M Dithriothrcilol (DTT) (0.4 ml)

Enzymatically digested/Sheared Herring Sperm DNA (0.2 ml) PEG 3350 (3.6 g) 100% Formamide (3.0 ml) Triton X-100 (0.5 ml)

Tween 20 (0.55 ml) 250 μl of probe stock solution (500 μg of probe)

The wash solution A had the following composition: 0.4 M guanidium isothiocvanale, 0.1 % Triton X- 100, 0.1 x SSC in deionized water. Wash solution B had the following composition: 0.1 % Triton X-100, 0.1 x SSC in deionized water.

Mounting solution was 0.1% 1 ,4 diphenylamine (anlifade) in 50% glycerυl (v/v) and nuclear stain Hoechst (#33258; 1 ug/ml). PEG is polyethylene glycol. SSC is 0.15 sodium citrate, 0.015 M sodium citrate. Ficoll/PVP is 5 g of Ficoll type 400 (polysucrose 400,000 molecular weight) plus 5 g of PVP (polyvinylpyrrolidone) diluted to a total volume of 100 ml with water. Sodium phosphate buffer is ph 6.8. The fluorescent signal was measured by a fluorescent microscope and compared to each other by intensity. An Olympus BII-2 microscope was used.

Fig. 1 is a sequence map of a portion of the "sense strand" human papilloma virus genome. The top strand is the sense strand; the bottom strand is the anli-sense strand. Base sequences in each strand were used to define the sequences of a series of oligonucleotides 25 bases in length ("25-mcrs") and separated by five bases along that strand. Two probe populations, A and B, were constructed such that each had all the probes whose sequences were defined by the sense strand (therefore those probes were capable of hybridizing to the anti-sense strand). Two probe populations, C and D, were constructed such that each had all the probes whose sequences were defined by the anti- sense strand.

The probes of populations A and B had probes equivalent to discrete regions 701- 71 1 of the sense strand with their Nucleotide Sequence ID No. as follows:

Probe SEO ID NO

700 SEQ ID NO: 1

701 SEQ ID NO: 2

702 SEQ ID NO: 3

703 SEQ ID NO: 4

704 SEQ ID NO: 5

705 SEQ ID NO: 6

706 SEQ ID NO: 7

707 SEQ ID NO: 8

708 SEQ ID NO: 9

709 SEQ ID NO: 10

710 SEQ ID NO: 11

711 SEQ ID NO: 12 ^"2&

SEQ ID NO: 13 is the sequence of the entire sense strand in Fig. 1.

The probes of populations C and D had probes equivalent to discrete regions 600- 1 1 of the antisense strand with their Nucleotide Sequence ID No. as follows:

Probe SEO ID NO

600 SEQ ID NO: 14

601 SEQ ID NO: 15

602 SEQ ID NO: 16

603 SEQ ID NO: 17

604 SEQ ID NO: 18

605 SEQ ID NO: 19

606 SEQ ID NO: 20

607 SEQ ID NO: 21

608 SEQ ID NO: 22

609 SEQ ID NO: 23

610 SEQ ID NO: 24

61 1 SEQ ID NO: 25

The regions complementary in base sequence lo discrete regions 700-71 1 and 600-

61 1 are discrete target regions. The discrete target regions form an overlapping pattern in

Figure 1, such that each len-base sequence at the end of a discrete target region on the

first strand is complementary in base sequence lo a ten-base sequence at an end of a

discrete target region on the other strand. Target sequences at the ends of the pattern,

however, i.e., those complementary in base sequence lo regions 600 and 61 1 , may only overlap one target sequence from the other strand.

Every probe in populations A and C had one tetramethylrhodamine moiety linked to its 5' end -nucleoside, but no tetramethylrhodamine or other reporter moiety linked to its 3'-end nucleoside. Every probe in populations B and D had a tetramethylrhodamine moiety linked to the nucleoside at its 3' end and a tetramethylrhodamine entity linked to the nucleoside at its 5' end. The probes in Population A and C were made using the procedure of Examples 1 and 4, thereby using Aminomodifier II to create the moiety linking the tetramethylrhodamine lo the 5' nucleoside.

The probes in population B and D were made using the methods of Examples 3 and 4, thereby using AminoModificr II to create the ifioicly linking the tetramethylrhodamine to the 5' nucleoside and using 3'-Amino-Modifier C7 CPG to create the moiety linking the tetramethylrhodamine lo the 3' nucleoside.

The results were obtained by visual observation using Ihc fluorescent microscope.

The results were as follows: Cells hybridized with population A gave a fluorescent signal similar in intensity to cells hybridized with population C. Cells hybridized with population B gave a fluorescent signal similar in intensity lo cells hybridized with population D. Cells hybridized with population B gave a fluorescent signal that was approximately twice as great as that obtained wilh population A. Cells hybridized with population D gave a fluorescent signal that was approximately twice as great as that obtained with population C. Cells hybridized with a combination of populations B and D gave a fluorescent signal that was approximately twice as great as cells hybridized with a combination of populations A and C.

The results show that the signal observed when both ends of a probe are labeled probe is about twice as great as that observed when only one end is labeled. The results also show that, when both ends of a probe are labeled, the signal obtained wilh a probe population that has probes for both target strands is about twice that obtained wilh a population that has probes for only a single strand.

2β

SEQUENCE LISTING GENERAL INFORMATION: (i) APPLICANT: APROGENEX, INC.

(ii) : TITLE OF INVENTION: Populations of Non-Adjacent Hybridization

Probes

(iii) NUMBER OF SEQUENCES: 25

(iv) CORRESPONDING ADDRESS:

(A) ADDRESSEE: Elraan, Wilf & Fried (B) STREET: 20 West Third Street

(C) CITY: Media

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19063

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette 5.25 inch

(B) COMPUTER: IBM PC Compatible

(C) OPERATING SYSTEM: PS-DOS/MS-DOS (D) SOFTWARE: WordPerfect for Windows

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(vii) Prior Application Data:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Elman, Gerry J

(B) REGISTRATION NUMBER: 24,404

(C) REFERENCE/DOCKET NUMBER: M19-050

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215 8⁽J2 9580

(B) TELEFAX: 215 892 9577

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERIΞICS: (A) LENGTH: 25 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TΛTGAAGAAA TTCCTATGGA TACAT 25

(3) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL II

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTTAGCACAΛ ACCCTAACAC AG AΛ 25

(4) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGCΛCACCCA TΛCCAGGGTC TCGCC 25

(5) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCΛCGCCTAG GΛTTΛTΛTAG TCGCΛ 25

(6) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CAACAGGTTA AΛGTTGTAGA CCCTG 25

(7) INFORMATION FOR SEQ Iϋ NO:6:

(i) SEQUENCE CHARACTERISES: (A) LENGTH: 25 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTAACCACTC CCACTAAACT TATTA 25

(8) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GATAATCCTG CATATGAAGG TATAG 25

(9) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHΛRACTF-RISICS:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY Linear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GATAATACΛT TATATTTTTC TΛGTΛ 25

(10) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AATAGTATTA ATATAGCTCC AGATC 25

(11) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTIO : SEQ ID NO: 10: TTTTTGGATA TAGTTGCTTT ACATΛ 25 (12) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCATTAACCT CTAGGCGTAC TGGCΛ 25 (13) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TΛCAGTAGAA TTGGTAATAA ACAAA 25

(14) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISES: (A) LENGTH: 450 (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ACACCTGCAG AAACTGGAGG GCATTTTACA CTTTCATCAT CCACTATTAG 50 TACACATAAT TΛTGAΛGAAΛ TTCCTATGGA TACATTTATT GTTAGCACAA 100 ACCCTAACAC AGTΛACTAGT AGCΛCΛCCCA TΛCCΛGGGTC TCGCCCAGTG 150 GCACGCCTAG GATTATATAG TCGCΛCAACA CAACAGGTTA AAGTT GTAGA 200 CCCTGCTTTT GTAACCACTC CCACTAAΛCT TATTACATAT GATΛATCCTG 250

CATATGAAGG TATAGATGTG GΛTΛΛTΛCΛT TΛTΛT TTTTC TAUTΛATGAT 300

AATΛGTATTA ATATAGCTCC AGΛTC'CTGΛC TTTTTGGATA TAGTTGCTTT 350

ACATAGGCCA GCATTAACCT CTAGGCGTAC TGGCΛTTAGG TACAGTAGAA 400

TTGGTAATAA ACAΛACACTA CGTΛCTCGTΛ GTGGAAAATC TATΛGGTGCT 450

(15) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISES: (A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: M

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TTCTACTGTA CCTAATGCCA GTACG 25

(16) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

AGGTTAATGC TGGCCTATGTAAAGC 25

(17) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TATCCAAAA GTCAGGATCT GGAGC 25

(18) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISES: (A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TAATACTATT ATCATTACTA GAAΛΛ 25

(19) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY Linear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ATGTATTATC CACATCTATA CCTTC 25

(20) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRI TION: SEQ ID NO: 19: CAGGATTATC ATΛTGTAATA AGTTT 25

(21) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nuclαic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GAGTGGTTAC AAAAGCAGGG TCTAC 25 (22) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISES: ( A ) LENGTH : 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TAACCTGTTG TGTTGTGCGA CTATA 25

(23) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: ilinear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CTAGGCGTGT CACTGGGCGA GACCC 25

(24) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TGGGTGTGCT ACTAGTTACT GTGT 25

(25) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISES:

(A) LENGTH: 25

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY Linear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TTGTGCTAAC AATAAΛTGTΛ TCCTΛT 25

(26) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISES: (A) LENGTH: 25 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY Linear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: TTTCTTCATA ATTATGTGTA CTAAT 25

Claims

1. A process of detecting a nucleic acid target molecule, said process comprising the steps of:

1) incubating, in a solution, a nucleic acid target molecule and a population of probe molecules, wherein the base sequences of the probe molecules will allow them lo hybridize lo discrete separated target regions along said target molecule, and

2) detecting probe molecules that have hybridized lo said target molecule, the separation between said target regions being 5 to 25 bases on average, wherein for each probe molecule, a first reporter moiety is linked cither directly or via a linker moiety indirectly lo the nucleoside al the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked cither directly or via a linker moiety indirectly lo the nucleoside al the 3' end of said probe molecule, wherein the sum of the molecular weights of said first reporter moiety and said first linker moiety docs not exceed 2000, and wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety docs not exceed 2000.

2. A process of Claim 1 wherein the length of each probe is between about 15 nucleotides and 40 nucleotides.

3. A process of Claim 2 wherein the length of each probe is about 25 nucleotides.

4. A process of Claim 1 wherein the the length of the linker is not more than 10 atoms.

5. A process of Claim 1 wherein the separation length between the target regions is 5 bases on average.

6. A process of Claim 1 wherein the target molecule is in a biological entity that is a cell or a virus.

7. A process of Claim 6 wherein the biological entity is a cell.

8. A process of Claim 7 wherein the cell is a eukaryotic cell.

9. A process of Claim 8 wherein the eukaryotic cell is a human cell.

10. A process of Claim 6 wherein the biological entity is a virus.

1 1. A process of Claim 6 wherein the biological entity is suspended in solution and not immobilized on a solid support.

12. A process of Claim 6 wherein the biological entity is immobilized on a solid support.

13. A process of Claim 6 wherein the biological entity is part of a tissue section taken from a multicellular organism or part of a histologic section taken from a multicellular organism.

14. A process of Claim I wherein the first and second reporter moieties arc fluorescent moieties.

15. A process of Claim 1 where the target molecule is a purified nucleic acids molecule.

16. A process of Claim 1 where the target molecule is DNA.

17. A process of Claim 1 where the target molecule is RNA.

18. A process of Claim 16 where the target molecule is viral DNA.

19. A process of Claim 18 wherein the viral DNA is human immunodeficiency virus DNA.

20. A process of Claim 18 wherein the viral DNA is human papilloma virus DNA.

21. A process of Claim 17 where the target molecule is viral RNA.

22. A process of Claim 21 wherein the target molecule is human immunodeficiency virus RNA.

23. A process of delecting a nucleic acid target molecule, said process comprising the steps of:

1) incubating, in a solution, a nucleic acid target molecule and a population of probe molecules, said target molecule comprised of a first strand and a second strand, said second strand complementary in base sequence to said first strand, said probe molecules comprising base sequences that allow them to hybridize both to discrete separated target regions on the first strands of said target molecule and discrete separated target regions on the second strand of said target molecule, and

2) detecting probe molecules that have hybridized lo a strand of said targcl molecule, the separation between said target regions on the first strand being 5 lo 25 bases on average, the separation between said target regions on the second strand being 5 to 25 bases on average, each ten-base sequence at the end of a discrete target region on the first strand strand being complementary in base sequence lo a ten-base sequence at an end of a discrete target region on the other strand, thereby creating an overlapping pattern of discrete target regions in the target molecule, provided Ihat target sequences at the ends of the pattern may overlap either one or two target sequences from the other strand, wherein for each probe molecule, a first reporter moiety is linked either directly or via a linker moiety indirectly to Ihe nucleoside at the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked cither directly or via a linker moiety indirectly lo the nucleoside al Ihe 3' end of said probe molecule, wherein the sum of the molecular weights of said first reporter moiety and said first linker moiety docs not exceed 2000, and wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety does not exceed 2000.

24. A process of Claim 23 wherein the length of each probe is about 25 nucleotides.

25. A process of Claim 23 wherein the separation length between the target regions is 5 bases on average.

26. A process of Claim 23 wherein the biological entity is a cell or a virus.

27. A process of Claim 26 wherein the biological entity is a cell.

28. A process of Claim 23 wherein the first and second reporter moieties arc fluorescent moieties.

29. A population of probe molecules, wherein the base sequences of the probe molecules will allow them lo hybridize to discrete separated target regions along a target molecule, the separation between the target regions being 5 lo 25 bases on average, wherein for each probe molecule, a first reporter moiety is linked either directly or via a linker moiety indirectly to the nucleoside al the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked either directly or via a linker moiety indirectly to the nucleoside at the 3' end of said probe molecule, Ato wherein the sum of Ihc molecular weights of said first reporter moiety and said first linker moiety does not exceed 2000, wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety does not exceed 2000, and said target molecule having a base sequence identical to or complementary to a nucleic acid molecule found in a biological entity that is a cell or virus.

30. A probe population of Claim 29 wherein the length of each probe is about 25 nucleotides.

31. A probe population of Claim 29 wherein the separation length between the target regions is 5 bases on average.

32. A probe population of Claim 29 wherein the biological entity is a human cell.

33. A probe population of Claim 29 wherein the first and second reporter moietiews are fluorescent moieties.

34. A probe population of Claim 29 wherein the biological entity is a virus capable of replicating in human cells.

35. A probe population of Claim 34 wherein the biological entity is human immunodeficiency virus.

36. A probe population of Claim 34 wherein the biological entity is human papilloma virus.

37. A kit for detecting a target molecule one having a base sequence identical to or complementary lo a nucleic acid molecule found in a biological entity that is a cell or virus, said kit comprising a probe population of Claim 29 and one or more reagents 4| selected from the group, a fixative and a chaotropic agent, said reagent intended for use in a solution for reacting said probe, population with said biological entity so that a hybrid molecule can form between a molecule of the probe population and said target molecule.