WO1996000234A1 - Centromere hybridization probes - Google Patents

Abstract

Probes and probe populations for detecting the centromeres of the X, and Y human chromosomes and for human chromosomes 1, 13, 18, and 21; use of those probe populations for chromosome identification; and kits designed to assist carrying out such processes.

Description

CENTROMERE 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, especially when dealing with intact cells, i.e., in in. situ procedures. li can, for example, be used to detect viruses, classify microorganisms, and detect genetic defects and gene expression. In many such cases, it is of interest to identify human chromosomes by hybridization procedures. The centromeric regions of human chromosomes offer potentially useful targets for hybridization because the centromeric base sequences are frequently repeated multiple times within a centromere. The use of centromere probes has been disclosed in European Patent Office applications EP 0511750A1 and EP 0473253A1. A probe specific for Chromosome X, and having the 25-nucleotide sequence TCGAAACGGG TATATGCTCA CGTAA was one of the probes reported in EP 0511750A1.

A high quality centromeric probe is one that results in well-defined and high intensity dots of detectable signal ("tight dots") when it hybridizes to a cell. It is not obvious in advance, however, that any given centromeric probe will be specific for a defined target and produce only tight dots. Indeed, for some centromeres, it is not obvious if any high quality probes can be constructed. Centromeric sequences (or sequences sufficiently similar so that they at least weakly hybridize to the probes) can also appear in non-centromeric regions or other centromeric regions. Furthermore, even if some probes are individually useful, it does not necessarily follow that a pool of several probes for the same centromeric region will also be useful. BRIEF SUMMARY OF THE INVENTION

The inventions are probes and probe populations for the centromeres of the human X chromosome, Y human chromosome, chromosome 1, chromosome 13, chromosome 18, and chromosome 21. Additionally, the inventions include processes for using those probes and probe populations for chromosome identification, as well as kits designed to assist carrying our such processes.

DETAILED DESCRIPTION

DEFINITIONS An "end-labelled probe" is one in which a reporter moiety is covalently linked to the nucleotide or nucleoside located at the 3' end, the 5' end, or both the 3' and 5' ends, of the oligonucleotide moiety of a probe. Linkage may be direct (as when an atom of the reporter moiety is directly linked to 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 that linker moiety.

"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).

"3SR" is an amplification system that uses oligonucleotide primers, a reverse transcriptase, DNA-dependent RNA polymerase, and RNase H (J.C. Guatelli et al, Proc. Nail. Acad. Sci. USA, 87, 1874 (1990).)

"TAS" is a transcription-based amplification system that uses oligonucleotide primers, a reverse transcriptase, and DNA-dependent RNA polymerase. (D.Y. Kwoh et al,

Proc. Nail. Acad. Sci. USA, 86, 1173 (1989))

"LCR", "LAR", "LAS", refer to "ligation chain reaction", "ligation amplification reaction", and "ligation-based amplification system" respectively, reactions which rely on a DNA ligase to join oligonucleotides that bind to a target (K.J. Barringer et al, Gene 89, 1 17 (1990); D.Y. Wu and R.B. Wallace, Genomics, 4, 560 (1989)).

"Qβ replicase" system uses that RNA bacteriophage enzyme to effect amplification. (P. M. Lizardi et al. Bio/Technology 6. 1197 (1988)). A "moiety" is part of a molecule. When a moiety links two molecular entities together, it may be referred to as a "linker moiety".

The term "dye" includes any molecule or molecular moiety that can be detected fluorimetrically or spectrophotometrically, especially though not necessarily in the visible range of wavelengths.

USES FOR THE PROBES

A probe specific for the centromeric region of the X chromosome is useful in determining whether a cell is of female origin (therefore having two X chromosomes) or male origin (therefore having only one X chromosome). Additionally, certain types of abnormal cells, such as aneuploid cells that have one or more extra chromosomes or have less than a normal complement of chromosomes, can be identified when such chromosomes involve the X chromosome, the Y chromosome, chromosome 1, chromosome 13, chromosome 18, or chromosome 21. Examples of such abnormalities are cells with two X and one Y chromosome, cells with one X and two Y chromosomes, cells with one X chromosome but no Y chromosome, or trisomies for chromosome 1 , chromosome 13, chromosome 18, or chromosome 21. Any centromeric probe is useful because, in conjunction with a second marker, it can be used to identify chromosomal translocation that results in either an abnormal linkage or unlinking of the two markers.

ASPECTS OF THE INVENTION

In one aspect, the current invention is an oligonucleotide probe population specific for a target that is the human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat, said probe population comprising at least three different probes, each having a probe nucleotide sequence different from the nucleotide sequences of the other two probes and not complementary to the nucleotide sequences of the other two probes, each of said three probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides (more preferably 20 to 30 nucleotides, most preferably 25 nucleotides) in said target (13 nucleotides is the approximate minimum number needed to achieve a stable hybrid molecule; increasing the lower limit from 13 to 20 nucleotides insures that there is a closer structural similarity to the sequences in the preferred group of 16 4 sequences below; 30 nucleotides is an approximate upper limit so that the probe does not deviate too much from the preferred sequences in the group of sixteen below), such that each of said three probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of sixteen probe nucleotide sequences,

TCGAAACGGG TATATGCTCA CGTAA and its complement, ATCGGGAATA TTTCCACAGA AAAAC and its complement, GAAGTTTCCC TTTGATAGCG CAGTT and its complement, TGACACACTT TTTCTACAAT GTGCA and its complement, ACTAAACACA AACAATCTGA GAAAG and its complement,

CGCAGAGATG AACCTGCCTT TGAGA and its complement, TGAAATATTC TTTTGGCAGA ATCTG and its complement, GACGAGAGAG AAGCATTGTC AGAAA and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said three probe nucleotide sequences that is less than 25 nucleotides being the same as one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing sixteen probe nucleotide sequences.

With two exceptions, each of the individual probes in the group of 16 specified sequences is itself an invention. The exceptions are the sequence TCGAAACGGG TATATGCTCA CGTAA and its complement, that sequence having already been disclosed as noted above. The phrase, "TCGAAACGGG TATATGCTCA CGTAA and its complement" refers to the nucleotide sequence "TCGAAACGGGTATATGCTCACGTAA", a sequence of 25 nucleotides and its complement, the sequence

"TTACGTGAGCATATACCCGTTTCGA" another sequence of 25 nucleotides. All sequences herein are written, left-to-right, in the 5' to 3' direction. In its complement, the T (or U) of a sequence is replaced by an A, an A by a T (or U), a G by a C, and a C by a G, and after all of the bases (T, A, G, C) have been replaced accordingly, the sequence of the nucleotides is reversed for purposes of representation so that, left to right, the nucleotides are in a 5' to 3' direction.

The use of the term "probe population" reflects the fact that, even if all the probe molecules that are added to the target cells in an assay are identical in structure, many

(thousands, millions, or even more) of those probe molecules will be in the volume of liquid in which the cells are suspended or immersed during the hybridization assay. That will be true regardless of whether or not the probe molecules all have the same nucleotide sequence. In many probe populations, not all the probe molecules will have the identical sequence. In such cases, the probe population will have "a population of nucleotide sequences"; i.e., two more different nucleotide sequences.

In another aspect, the current invention is an oligonucleotide probe population specific for a target that is the human Y-chromosome specific repetitive DNA Family (DYZ1) sequence, said probe population comprising at least three different probes, each having a probe nucleotide sequence different from the nucleotide sequences of the other two probes and not complementary to the nucleotide sequences of the other two probes, each of said three probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides (more preferably 20 to 30 nucleotides, most preferably 25 nucleotides) in said target, such that each of said three probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of sixteen probe nucleotide sequences, GAGCCCTTTC AATTTGAGTC CATTC and its complement,

GAGTCGATTT TATTGCATTA GATTC and its complement, AGAGGACATT CCATTCCAAT GCATT and its complement, TTCTATTCCC TTCTACTGCA TACAA and its complement, TTGGATTACT TTCCATTCGA TTACA and its complement, TCTATTCTAT TACATAACTT TCCAT and its complement,

ACAGTCCATT CCAATAGATT CCATT and its complement, ACTAGACCAT TCCAAACCAG TCCAT and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said three probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing sixteen probe nucleotide sequences.

Each of the individual probes in the group of 16 specified sequences for the Y chromosome is itself an invention. In a third aspect, the invention is an oligonucleotide probe population specific for a target thai is the human alphoid repetitive DNA LI.84 mapping to chromosome 18, said probe population comprising at least two different probes whose nucleotide sequences are partially complementary to each other, each having a probe nucleotide sequence different from the nucleotide sequence of the other and not completely complementary to the sequence of the other probe, each of said two probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides (more preferably 20 to 30 nucleotides, most preferably 25 nucleotides) in said target, such that each of said two probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of four nucleotide sequences,

TCTTAGTGTG AGTACACACA TCTCA and its complement, CTCACACTAA GAGAATTGAA CCACC and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said two probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing four probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing four probe nucleotide sequences.

Each of the individual probes in the group of four specified sequences for chromosome 18 is itself an invention. In a fourth aspect, the invention is an oligonucleotide probe population specific for a target that is the human chromosome 1 III DNA fragment, 5' end, 132 BP repeat, said probe population comprising at least two different probes, each having a probe nucleotide sequence different from the nucleotide sequence of the other and not completely complementary to the nucleotide sequence of the other probe, each of said two probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides (more preferably 20 to 30 nucleotides, most preferably 25 nucleotides) in said target, such that each of said two probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of four nucleotide sequences,

ATCATCATCA AATGGAATCA AAAATA and its complement, AATTCATTTG AAGACAATTC CATTCA and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing four probe nucleotide sequences.

Each of the individual probes in the group of four specified sequences for chromosome 1 is itself an invention.

In a fifth aspect, the invention is an oligonucleotide probe population specific for a target that is human alphoid repetitive DNA LI.26 mapping to chromosomes 13 and 21 , said probe population comprising at least three different probes, each having a probe nucleotide sequence different from the nucleotide sequence of the other two probes and not completely complementary to the nucleotide sequence of the other two probe, each of said three probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, such that each of said probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of six nucleotide sequences, AATTCAAATA AAAGGTAGAC AGCAG and its complement, CCCATAAAAA CGAGACAGAA GGATT and its complement, GATATTTAGA TTGCTTTAAC GATAT and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA. each of said probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing six probe nucleotide sequences, such that in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing six probe sequences. Each of the individual probes in the group of six specified sequences for

Chromosomes 13 and 21 is itself an invention.

In the foregoing aspects of the invention where there are 16 sequences in the group, optimally as many as eight, of the sequences can be selected to appear on probes in the population, providing each selected sequence is not complementary to one of the other selected sequences. If the selected sequences are complementary to each other, the populations would still be useful but would be less than optimal, because some molecules in the probe population would hybridize to other molecules in the probe population and not be available for hybridization to the target. Nevertheless it is possible to have both a probe nucleotide sequence and its complement in a probe population and for it to be of some, as there will be some hybridization to both strands of the target.

For some of the probe populations, such as "An oligonucleotide probe population specific for a target that is the human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat", there is a limitation of the type, "such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing sixteen probe nucleotide sequences." That limitation guarantees that the preferred sixteen probe sequences will account for at least half of the sequences in the population specific for the target and thereby guarantee that they will make a significant contribution to the quality (i.e., specificity) of the probe population. An illustration of how one determines whether, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing sixteen probe nucleotide sequences, is the following; If there are five different probe nucleotide sequences in the probe population specific for the target, three of which are 25 nucleotides in length and a member of the group of 16 probe sequences, two of which are 40 nucleotides in length and not members of the group of 16 probe sequences, then the sum of the lengths of the probe nucleotides is 155 (i.e., 3 times 25 plus 2 times 40) and the contribution of the preferred 16 nucleotide sequences is 75 (i.e., 3 times 25).

The target may be a centromere region or an amplification product thereof, such as the product of PCR, ligation chain reaction, ligation amplification reaction, ligation-based amplification system. Preferably, the centromeric probes each have a size that does not exceed 40 nucleotides.

In another aspect, the invention is a process of using the probes and/or probe populations that are aspects of this invention to detect a nucleic acid target molecule in a human cell or chromosome spread, which process comprises the steps of (1) co-incubating the human cell or chromosome spread with an oligonucleotide probe or probe population (described herein) that is specific for a target is a region of centromere of human chromosome 1 or 13 or 18 or 21 or the X or Y human chromosome,

(2) detecting the oligonucleotide probe molecules that arc hybridized to the target molecules inside the cell or in the chromosome spread; such that step (1) is performed under conditions that allow the oligonucleotide probe to hybridize to the target molecule.

In another aspect, the invention is a kit for analyzing human chromosomes, said kit comprising a probe population (or probe) that is an aspect of the current invention and that will detect a centromeric region of either chromosome 1, chromosome 13, chromosome 18, chromosome 21, chromosome X, or chromosome Y, said kit further comprising either instructions for using said probe population to detect a human chromosome or an indication that a use for said probe population is to detect a human chromosome or centromere.

A probe population that is specific for a particular centromeric region can retain its specificity even if it is mixed with other DNA molecules. For example, the probe 10 population may be mixed in with an excess of DNA that does not bind specifically to the DNA of the target cells of interest. That would be the case where the target cells are human cells and there is an excess of human Cot DNA or salmon sperm DNA, the reason for human Cot DNA or the salmon DNA being to block the probe population from non- specific binding sites in the target cells.

Another example of a probe population retaining its specificity even when mixed with other DNA molecules is when there are two (or more) probe populations mixed together. If the two populations are labeled with distinguishable reporter groups, e.g., fluorescent dyes that emit different colors, then both populations retain their specificity. Simultaneous use of multiple reporter groups has been disclosed in PCT application

PCT/US/03582; P.M. Nederloff et al., Cylometrv. vol 10, pp 20-27 ( 1988).

A probe population may also retain its specificity for a particular centromeric region even if one or more of the probe molecules each have two (or more) different types of DNA sequences. For example, part of a probe molecule may be a nucleotide sequence specific for a particular target region (that being the "probe nucleotide sequence") and a second nucleotide sequence (referred to as a "reporter nucleotide sequence") that does not hybridize to the target DNA, but rather exists as a single-stranded "tail" in solution after the probe nucleotide sequence hybridizes to a target sequence. The reporter nucleotide sequence can function as part of the reporter system. For example, the reporter nucleotide sequence may act as a target for other nucleic acid molecules that are linked to fluorescent moieties or biotin moieties further bindable to streptavidin covalently linked to fluorescent moieties) (e.g., Schneider et al., PCT application PCT/US 86/02719). Alternatively, the reporter nucleotide sequence may act as a target for other nucleic acid molecules that , or act as a promoter for a polymerase (such as RNA polymerase or a DNA polymerase) in an amplification system (e.g., Loewy et al, EP application 8931 1834.9; M. S. Urdea, PCT application, PCT/US91/00213).

The probes of this invention are also useful probes when the target is purified DNA.

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 in whole or in part by heat or other means before the hybridization step takes place. The hybridization can take place with the target in solution phase or immobilized on a solid support (e.g., nitrocellulose paper for DNA, nylon for RNA) by well-established procedures.

The hybridizations vary considerably, depending in part on the level of specificity desired. Some examples are the Southern Blot procedure ( J. Mol. Biol., 98, 503-517

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

It is understood that a hybrid molecule comprising a probe molecule and a target molecule is formed because the probe has a nucleotide sequence complementary to a nucleotide sequence of the target molecule (e.g., an adenine complementary to either a uracii or a thymine in the other strand, a guanine complementary to a cytosine in the other strand). It is not necessary, however, that the entire nucleotide (or nuclcoside) sequence of the probe be complementary to the entire nucleotide sequence of the target.

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 the bases adenosine, uridine, thymidine, guanine, cytosine, or any natural or artificial chemical derivatives thereof.

Probes that are RNA can be made using a DNA synthesizer from Applied Biosystems, Inc., Foster City, CA, USA, with RNA reagents supplied by that company.

Probes 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 chemiluminescers 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, ¹²⁵I and ^, C. Other reporter moieties are ones that are enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (sec 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 Photobiotin™ labeling of probes are detected after hybridization using fluorescent, enzymatic or colloidal gold conjugates of avidin/strcptavidin. Nucleic acids may also be labeled with immunodetectable reporter moieties such as digoxigenin detectable with anti-digoxigenin antibodies.

One preferred 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.

When the reporter moiety is a fluorescent dye, probes bound to the target are detected by exposing the target-bound probe to light at a wavelength that is absorbed by the dye, and detecting the light emitted by the dye moiety.

Another preferred reporter group is an enzyme such as alkaline phosphatase or horse radish peroxidase. If such enzymes are linked to the probe (either covalently prior to hybridization or noncovalently after hybridization, possibly as part of a complex involving other moieties such as biotin and streptavidin, they can be used to convert nondetectable (under the conditions of the assay) precursors to detectable products that serve as a signal that hybridization has taken place.

An example of a reporter group that can be detected by a nonfluorescent method is biotin, which can be detected on the basis of its ability to bind to second compound, streptavidin, which in turn can be linked to enzymes such as alkaline phosphatase or horse radish peroxidase that are detectable on the basis of their ability to react with a substrate. Moieties that participate in chemiluminescent reactions {e.g., 3-aminophthalhydrazide ("luminol"), 4-methoxy-4-(3-phosphatephenyl)-spiro-( 1 ,2-dioxetane-3,2'-adamantane) disodium salt, and moieties that react with antibodies are also among the possibilities for reporter moieties.

A probe should normally be at least about 13 bases long for specific hybridization to take place.

TARGETS IN CELLS. TISSUE. AND FLUIDS The hybridization assay can also be done for targets in biological entities in liquid suspension, for targets in cells on slides or other solid supports, for targets in tissue culture cells, and for targets 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, tesles, cervix, bone marrow, and brain) or cells present in membranes lining various tracts, conduits and cavities (such as the gastrointestinal tract, urinary tract, vas deferens, 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 IS IN A BIOLOGICAL ENTITY Two useful summaries of possible hybridization conditions are in PCT International

Patent Applications with publication numbers WO 90/02173 and WO 90/02204, both of them applications of Research Development Corp.

In situ hybridization allows the detection of RNA or DNA sequences within individual cells. It can detect as few as 1-5 target molecules per cell in as little as 2-4 hours. (PCT Applications WO 90/02173 and WO 90/02204) It also allows for the simultaneous detection of more than one different polynucleotide sequence in an individual cell. It also allows detection of proteins and polynucleotides in the same cell.

Many different hybridization conditions (solvent composition, temperature, time) are possible. The ones mentioned below are 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% formamidc, 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 (pH 7.4), about 100 micrograms (ug)/milliliter (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 raM vanadyl ribonucleoside complexes.

All percentages for liquids are 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 50°-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 formamide, urea, thiocyanate, guanidine, trichloroacetate, tetramethylamine, perchlorate, and sodium iodide. Any buffer which maintains pH at least between 7.0 and 8.0 is preferred.

The pore forming agent is for instance, a detergent such as Brij 35 (23 lauryl ether), Brij 58 (20 cetyl ether), sodium dodecyl sulfate, CHAPS™ ((3-[3- cholamidopropyl)-dimethylammonio]-l -propane sulfonate), Triton X-100 (a polyoxyethylene ether sold by Sigma Chemical Company, St. Louis, MO) or Tween

(polyoxyethylene sorbitan sold by Sigma Chemical Co.). Depending on the location of the target biopolymer, the pore-forming agent is chosen to 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 entry through the plasma membrane but not the nuclear membrane. Alternatively, sodium desoxycholate will allow probes to traverse the nuclear membrane.

Hybrid stabilizing agents such as salts of mono- and di-valent cations are included in the hybridization solution to promote formation of hydrogen bonds between complementary nucleotide sequences of the probe and its target biopolymer. Preferably sodium chloride at a concentration from .15 M to 1 M is used. Many fixative agents may also serve as hybrid stabilizing agents and vice versa. In order to prevent non-specific binding of nucleic acid probes, nucleic acids unrelated to 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 subcellular structures as close as possible to the fresh untreated cells is chosen as optimal for each step.

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

MOUNTING BIOLOGICAL ENTITIES/TISSUES

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 arc 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 quantitation procedure used for hybridization detection. Some filter materials are not uniformly thick and, thus, shrinking and swelling during in situ hybridization procedures is not uniform. In addition, some supports which autofluoresce 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-noise ratios and can be treated to 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, neutral buffered formalin, 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 chromate, 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 the following characteristics: the effect is reversible, the cellular (or viral) morphology of interest is maintained, the antigenicity of desired cellular constituents is maintained, the nucleic acids arc retained in the appropriate location in the cell, the nucleic acids are not modified in such a way that 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 to 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 are selected from the group consisting of ethanol, ethanol-acetic acid, methanol, and methanol-acetone which fixatives afford the highest hybridization efficiency with good preservation of cellular morphology.

Fixatives for practicing the present invention include 95% ethanol/5% acetic acid for IIL-60 and normal bone marrow cells, 75% ethanol/20% acetic acid for K562 and normal peripheral blood cells, 50% methanol/50% acetone for fibroblast cells and normal bone marrow cells, and 10% formaldehyde/90% methanol for cardiac muscle tissue. 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, glutaraldehyde 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 covalently modify nucleic acids preventing later hybrid formation. STORAGE OF BIOLOGICAL ENTITIES/TISSUES

After fixation, microscope slides containing cells may be stored air dried at room temperature for up to 18 months, in cold (4°C) 70% ethanol in water for 6-12 months, or in paraplast for up to two years. If specimens are handled under RNase-free conditions, they can be dehydrated in graded alcohols and stored for at least 18 months at room temperature.

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

HYBRIDIZATION IN SOLID TISSUE

In a typical procedure, four 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 min. at room temperature.

Hybridization then proceeds using procedures described elsewhere in this document.

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 are placed into a single cell suspension of about lθ θ⁶ 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 may be carried out in the same solution which effects fixation. This solution may contain both a fixative and a chaotropic agent such as formamide. Also included in this solution is a hybrid stabilizing agent such as concentrated sodium or lithium chloride or ammonium acetate solution, a buffer, low molecular weight DNA and/or ribosomal RNA (sized to about 50 bases) to diminish non-specific binding, and a pore forming agent to facilitate probe entry into the cells. Nuclease inhibitors such as vanadyl ribonucleosidc complexes may also be included. To the hybridization solution is added a probe (or probes), to hybridize with a target polynucleotide.

The one-step procedure is a means of carrying out the fixation, prehybridization, hybridization and detection steps normally associated with in situ hybridization procedures all in one step. By modifying the components of this "one-step" solution, a convenient temperature 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 may also fix the cells. This fixation may be 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 may be placed into a PBS solution (maintained to cellular osmolality with bovine serum albumin (BSA) at a concentration of 10⁵ to 10⁶ cells per ml. Cells in suspension may be fixed and processed at a later time, fixed and processed immediately, or not fixed and processed in the i situ hybridization system of the present invention.

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

The fixative is one which has been found to be optimal for the particular cell type being assayed (eg., there may be 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 application PCT/US89/03582) and U.S. patent 5,225,236 (Bresser et al.) The hybridization cocktail contains a denaturing agent, usually formamide 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 denaturant in either an additive or synergistic fashion. The hybridization cocktail may be constructed to preferentially allow only the formation of RNA-RNA or RNA-DNA hybrids. This is accomplished by adjusting the concentration of the denaturing agents along with the concentration of salts (primarily monovalent 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 "fixes" the morphology of the cells/tissues.

KITS

In addition to comprising a probe population (or probe) that is an aspect of the current invention, a kit may comprise one more reagents 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 molecule in the biological entity.

For example, a kit could include a solution containing a fixation/hybridization cocktail and one or more labeled probes. This solution could, for example, contain 0-40% ethanol/methanol, 25-40% formamide, 0-10% formaldehyde, 0.1-1.5 M LiCl, 0.05-0.5 M Tris-acetate (pH 7-8), 0.05%-0.15% Triton X-100, 20 ug/ml-200 ug/ml of a non-specific nucleic acid which does not react with the probe(s), and 0.1 ug/ml to 10 ug/ml of single stranded probes directly labeled with a reporter molecule.

Alternatively, the kit could include concentrated stock solution(s) to 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 to affix cells to said support, or a device to assist with any incubations or washings of the specimens, a photographic film or emulsion with which to record results of assays carried out with the present invention.

FLUORESCENT MEASUREMENTS Fluorescent measurements can be made using a fluorescent microscope such as an

Olympus BH10 microscope with fluorescent capabilities. Fluorescent measurements can also be made on a flow cytometer, such as a FACSTAR^1M made by Becton Dickinson.

CHARACTERIZATION OF RESULTS UNDER THE MICROSCOPE

A "tight dot" is one that is essentially symmetrical (circular perimeter) and small compared to the total size of the cell. In a perfect assay system, all dots observed with control cells (e.g., normal cells) are tight, there is one dot for each target region, and there is no other signal other than that created by the tight dots. A "split dot" is one that appears as two tight dots that are either slightly overlapping or just touching each other. Split dots are the result of a probe hybridizing to two regions, one normally the desired target region. Split dots, if they appear in normal or control cells are not a desirable aspect of an assay. They limit the ability of the assay to detect chromosomal aberrations. Split dots, when seen, will not necessarily be seen in all cells: In some cell orientations, one dot may be hidden behind the other along the viewer's line of sight.

A "diffuse signal" is one that is symmetrical and shows a bright region bordering one or more less bright regions. Diffuse dots are caused by the probe binding to nontarget regions of a cell's chromosomes at a lesser intensity than it its binding to its target region. As with split dots, the appearance of the diffuse signal can vary from cell to cell depending on the cell's orientation with respect to the viewer's line of sight. Diffuse dots are not a desirable feature of an assay.

The terms "tight dot", "split dot", and "diffuse signal" can be applied not only to targets in fixed cells, but also to chromosome spreads obtained by lysing the cells. The specificity of each X chromosome oligonucleotide probe can be determined by hybridizing it to human female cells (XX) and human male cells (XY). An oligonucleotide is then considered specific if it gave two tight dots in the female cells (or chromosome spreads from them), one tight dot in the male cells (or chromosome spreads from them), and no other signal.

The specificity of each Y chromosome oligonucleotide probe can be determined by hybridizing it to human female cells (XX) and human male cells (XY). An oligonucleotide is then considered specific if it gave no tight dots in the female cells (or chromosome spreads), one tight dot in the male cells (or chromosome spreads), and no other signal.

The specificity of each chromosome 18 oligonucleotide probe can be determined by hybridizing it to normal human female cells (or chromosome spreads from them) and those with a trisomy for chromosome 18. An oligonucleotide is then considered specific if it gave two tight dots in the normal cells (or chromosome spreads), three tight dots in the trisomic cells or spreads, and no other signal.

The specificity of each chromosome 1 oligonucleotide probe can be determined by hybridizing it to normal human female cells, chromosome spreads, or metaphase spreads. An oligonucleotide is considered specific if it gave two tight dots in the normal cells or their chromosome spreads, and no other signal.

The specificity of each chromosome 13/21 oligonucleotide probe (a probe that will hybridize to either the centromere of chromosome 13 or the centromere of chromosome 21) can be determined by hybridizing it to normal or trisomic human cells, chromosome spreads, or metaphase chromosome spreads. An oligonucleotide is then considered specific if the only signal it generated was four tight dots in normal cells or spreads, or five tight dots in cells or chromosome spreads trisomic for either chromosome 13 or 21.

ADDITIONAL USEFUL REAGENTS AND SOLUTIONS

Useful reagents and solutions for performing hybridization include 0.0025% Evans Blue and or 10% dodecyl alcohol 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; if a probe with a promoter region is used, it may be advantageous to add a compound selected from the group, dimethyl sulfoxide, an alcohol, an aliphatic alkane, an alkene, a cyclodextrin, a fatty acid ester, an amide or lactam, and an organic saline, to the solution containing the amplifying polymerase when it is added to the cells containing the hybridized probe; 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 85 °C for 15 min

(or at 90°C for 5 min followed by 42°C for 30 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 the probes are labelled at both ends, such as with tetramethylrhodamine via a linker generated by Aminolink 2 linker (Applied Biosystems, Inc. Foster City, U.S. A.) at the probe's 5' end and via, at the probe's 3' end, a linker generated by 3'-Aminomodifier C7 CPG { ( 1 -dimethoxytrityloxy-3-fluorenylmethoxycarbonylaminohexan-2-methylsuccinoyl)- long chain amino moiety linked to controlled pore glass, sold by Glen Research, Sterling, VA), it may be advisable to have the target regions spaced at least five bases apart along the centromere target.

PROCEDURES USED FOR THE EXAMPLES: SYNTHESIS OF PROBES

Except as noted, the synthesis procedure was as follows, or with minor variations thereof:

Aminolink 2 {6-trifluoroacetylamino)hexyl-(2-cyanoethyl)-(N,N diisopropyl)- phosphoramidite} was added to the 5' end of each oligonucleotide by standard phosphoroamidite chemistry using an Applied Biosystems Inc. DNA Synthesizer except that after the oligonucleotide was synthesized, Aminolink 2 was reacted with the oligonucleotide's 5' hydroxyl group in the same manner as a protected phosphoramidile nucleoside would react with that hydroxyl during normal oligonucleotide synthesis by the DNA synthesizer. Continuing with the DNA synthesizer as in the normal oligonucleotide synthesis cycle, all the protecting groups (including the trifluorocarbonyl group on the Aminolink 2) were removed and the oligonucleotide was cleaved from the DNA Synthesizer's column. Tetramethylrhodamine was then added to the Aminolink by reacting TAMRA {5-(and-6)-carboxytetramethylrhodamine succinimidyl ester, sold by Molecular Probes, Cat. No. 1171 } in carbonate buffer pH 9.0 at 23 °C for 3 to 12 hours) and precipitating the labelled oligonucleotide by adding 0.1 vol of 3M sodium acetate (pH 5.2) and 2.5 x total volume of cold ethanol, and incubating the resulting mixture at -20°C for 30 min. After resuspension in water, the oligonucleotide is further purified by Sephadex G-50 exclusion chromatography and reversed phase HPLC, ethanol precipitation of the desired fractions, and purification as regard size on a 20% polyacrylamide gel.

When FITC was used instead of tetramethylrhodamine then the following changes were made in the above procedure: Oligonucleotides linked to Aminolink were reacted fluorescein isothiocyanate (FITC) sold by Molecular Probes cat. No. F-143, in carbonate buffer pH 9.0 at 23 °C for 3 to 12 hours.

PROCEDURES FOLLOWED IN THE EXAMPLES: PREPARATION OF CELLS FOR HYBRIDIZATION AND HYBRIDIZATION CONDITIONS:

Except as noted, cells were prepared for hybridization and hybridized with probes essentially as follows, or with minor variations: Cells were grown to confluence in 5% CO₂, then rinsed in IX PBS. To the cells were added 0.25% trypsin in 0.02 M EDTA. 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 ethanol: methanol (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 85°C for 15 min after which the cells were washed once with wash solution A and five times with wash solution B before being mounted in mounting solution.

The hybridization solution was made according to the following formula:

500X Ficoll/PVP (0.3ml) 4M Guanidine thiocyanate (1.0 ml)

0.5 M EDTA buffer (pH 8.0)

30 X SSC (1.66 ml)

1 M sodium phosphate buffer (0.5 ml)

5M Dithriothreitol (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) Probe at a concentration of 300 ug/10 ml in the hybridization solution.

Where less than 10 ml of hybridization solution was needed for a particular probe, which was normally the case, the amounts of all components were scaled down proportionately.

The wash solution A had the following composition: 0.4 M guanidium isothiocyanate, 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 (antifade) in 50% glycerol (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 pll 6.8.

The fluorescent signal was measured by a fluorescent microscope and compared to each other by intensity. An Olympus BH-2 microscope was used.

PROCEDURES FOLLOWED IN THE EXAMPLES: PREPARATION OF CHROMOSOME SPREADS FOR HYBRIDIZATION AND HYBRIDIZATION CONDITIONS

White blood cells were stimulated with mitogen (phytohemagglutinin at 1 ug/ml) to divide; while dividing, the cells were arrested in metaphase with colchicine (0.01 ug/ml) which disrupts microtubules. Cells were suspended in hypotonic solution (0.075 M KC1 for 15 to 30 min then resuspended in methanol :acetic acid (3: 1) and dropped onto glass slides where they lysed. NUCLEOTIDE SEQUENCES OF CENTROMERE REGIONS REFERRED TO IN THE EXAMPLES

"Human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat", Waye. J.S. and Willard, H.F., Nucleic Acids Res., vol 13, pages 2731-2743 (1985), Gen Bank accession number X02418, had the following sequence (each line, 5' to 3', left to right, the number to the far right of each line denoting the cumulative number of nucleotides from the beginning of the sequence to the nucleotide at the right end of that line):

GATCCGCAAG GGATATTTGG ACCTCTTTGA AGGTTTCGTT GGAAACGGGA 50

TAATCTTCAC CTAAAAGCTA AACGGAAGCA TTCTCAGAAA CTTCTTTGGG 100

ATGTTTGCAT TCACCTCACA GAGTTGAAGT TTCCCTTTGA TAGCGCAGTT 150

TGACACACTT TTTCTACAAT GTGCAAGTGG CTATTTAGCG GGCTTGGAGG 200 ACTGTGTTGG AAAAGGAAAT ATCTTCTCCT AAAAACGACA TAGAAGCATT 250

CTCAGAAACT GCTCTGTGAT GATTGCATTC AACTCCCAGA GTTGAACATT 300

CCTTTTGATA GAGCAGTTTG CAAACACTCT TTTTGTAGAA TCTGCAAGTG 350

GAGATTTGGA CCGCTTTGAG GCCTGTGGTA GTGAAGGAAA GAACTTCATA 400

TAAAAACCAG ACGGTAGCAC TCTCAGAAAA TTCTTTGTGA CGATGGAGTT 450 TAACTCAGGG AGCTGAACAT TCGTTATGAT GGAGCAGATT CCAAACACAC 500

GTTTTGTAGA ATCTGCGAGG GGATATTTGG ACTCTCTCTG AGGATTTCGT 550

TGGAAACGGG ATCACCTTCC CATAACTGAA CGGAAAGCAA ACTCAGAACA 600

TTCTTTGTGA TGTTTGTATT CAACTCACAG AGTTGAACCT TCCTTTGATA 650

GTTCAGGTTT GCAACACCCT TGTAGTAGAA TCTGCAAGTG TATATTTTGA 700 CCACTTTGTA GCCTTCGTTT GAAACGTCTA TATCTTCACA TCAAACCTAG 750

ACAGAAGCAT TCTCAGAAAG TTTTCTGCGA TGACTGCATT CCACTCACAG 800

AGTTGAACAA TCCTTTGTGA TGGAGCAGTT TTGAAACCCT CTTTCTTTGG 850

AATCTGCAAG GGGATATGTG GACCTCTTTG AAGATTTCAC TGGAAACGGG 900

ATCATCTTCA CATAAAAACT AAACAGAAGC ATTCTCGGAA ACTATTTTGT 950 GATGTTTGTA TTCAACTCCC AGAGTTGAAC TTTCCTTTTG AAAGAGCAGC 1000

TATGAAACAC TCTTTTTCGA GAATCTGCAA GTGGACGTTT GGAGGGCTTT 1050

GAGGCTGTGG TGGAAAAGGA AATATCTTCA CACAAAAACC AGATAGAAGC 1100

ATTCTCAGAA ACGACTTTGT GAGGATGGCA TTCAACTACT GGAGTTGACA 1150

ATCCTATTGA TAGAGCAGAT TGGAATCACT CTTTTTGTAG AATCTGCAAA 1200 TGGAGATTTG GACTGCTTTG AGGCCTACGG TGGTACAGGA AGGAACTTCA 1250

TATAAAAGGC AAACGGAAGC ATTCTCAGAA TATTCTTTGT GATGATGGAG 1300 TTTCACTCAC AGAGCTGAAC ATGCCTTTTG ATGGAGCAGT TTCCAAATAC 1350

ACTTTTGGTA GAATCTGCAG GTGGATATTT GGAGCTCTCT GAGGATTTCG 1400

TTGGAAACGG GAATAATTTC CCATAACTAA ACACAAACAA TCTGAGAAAG 1450

TTCTTCATGA TGAATGCATT TAACTCGCAG AGATGAACCT GCCTTTGAGA 1500 GTTCAGGTTC GAAACACTCT TTCTGTAGAA TCTGCAAGTG GATATTTGGA 1550

CCACTGGGTG CCTTCGTTCG AAACGGGTAT ATGCTCACGT AAAAACTAAA 1600

GAGAAGCATT CTCAGAAACT TCTGAGTGAT GATTGCATTC AAGTCACACA 1650

GTTGAACCCT CCTTTTGATG GAGCAGTTTT GAAACTGTCT TTTTGTAGAA 1700

TCTGTAAGTG GATACGTGGA CCTCTTTGAA GATTTCTTTG GAATCGGGAA 1750 TATTTCCACA GAAAAACTAA ACTGAAGCAT TCTCAGAAAC TGCTTTGTGG 1800

TGTTTGTGTT CGAGCCGCAG AGTTTAACAT TGCTTTTCAT AGAGCAGTTT 1850

TGAAATATTC TTTTGGCAGA ATCTGCAAGT GGACATTTGG AGCGCTTTCA 1900

GGCCTGTGGT GGAAAAGCCT GAAAGCCTTT TCGCTTTATC TTCACAGAAA 1950

GACGAGAGAG AAGCATTGTC AGAAACTTCT TTGTGATGAT TGCATTCAAC 2000 TCACAGAGTT GAAGATTCCT TTTGAAACAG CAGTTTCGAA ACACTCTTTC 2050

TGTGG 2055

The "Human Y-Chromosome specific repetitive DNA Family (DYZ1) Sequence", See Y. Nakahari et al, Nucleic Acids Res., vol 14, pp7569-7580 (1986), a 3564 base pair repeat, Gen Bank Accession number X06228, had the following sequence:

TTCCATTCCA TTCCAATCCA TTCCTTTCCT TTCGCTTGCA TTCCATTCTA 50

TTCCCTTCTA CTGCATACAA TTTCACTCCA TTCGTTCCC TTCCATTCAA 100

TTCCATTCCA TTCAATTCCA TTCCATTTGT TTCCATTCTC TTCGATTCCA 150

TTTCTTTATA TTCCATGCCA TTCGATTCCA TTCTATTGGA TTGCATTACA 200

TACGTGTTCA TTCCATTCCA GACCATTCCA TTTGACTCCA TTCCTTTCGA 250 GCCCTTTCAA TTTGAGTCCA TTCCTTTCCA GTCCATTTCA CTCCAGTCCA 300

TTACTATCCA TTCCATACCA TTCCATCCCA TTCCATTCCA TTCCATTCCA 350

TTCCATTCCA TTGCATTCCA TTCCATTCCA TTCCATTCCA TTCCATTCCA 400

TTGCACTGCA CTCCATTCCA TTACATTCTA CTCTATCTGA GTCGATTTTA 450

TTGCATTAGA TTCTATTCCA TTGGATTACT TTCCATTCGA TTACATTCCA 500 TTCATGTACA TTCCATTCCA GTCAATTACA TTCGAGTTCA TTACATTACA 550

TTCCAGTATA TTCCATTGTA TTCGATCCCA TTCCTTTCAA TTCCATTTCA 600

TTCGACTCCA TTATATTCGA TTCCATTCCA CTCGAATCCA TTCCATTAGA 650

GGACATTCCA TTCCAATGCA TTCCATTCCA TTCCATAGCA TTCCATTGCA 700

TTCGATTCCA TTCCATTTGA TGCCATTCCA TTTGATGCCA TTCCATGACA 750 TTCCATTCCA TTCGAGTCCG TTCCGTTCCA ATTCATTGCA TTCCGTTTCA 800

TGAAATTCGA GTCCTTTCCA GTACATTTCA TTCCAATCCC ATCCAATCCA 850

ATCTACTCCA TTCAATTCCT TTCCATTCCA TTTGATTTGA TTCCATTGAT 900

TTGATTCCAT TCAGTTTGAT TCCATTCCGT GAAATTTCGT TCCATTCTAT 950

TCTATTACAT AACTTTCCAT TCAATTCCAT TCCATTTCAT TTCAGTCCAT 1000 TCGCTTCCTT TCCTTTCGAT TCAATTCCAT TTGATTCCAC TCCATTCTAT 1050

GCAATTTCAT TCCAATCGAT TCAATTCCAT TCGATGACAT TCCTTTCGTT 1100

TCCATTCCAT TCGAGTCCAT TCAATTTGAG CATTCGTGTC CATTCTATTC 1150

GAGTCCATTC CATTACCGTC TATTCTATTC CCTTCCATTC CTGTTGATTC 1200

AATTTCATTC CCTTCCATTC GATTCCTTTC CATTCGATTC CATTCCTTTC 1250 CATTCCATTC CATTCGTTCC CATTCCATGT GATTTCATTC CATTCCAGTC 1300

CATTATATTC GAGTCCACTC CACTCCATTC TATTACATTC AATTCCTTTT 1350

GAGTCCGTTC CATAACACTC CATTCATTTC GATTCCATTT CTTGACAGTT 1400

TTCTTCCATT TTATTCCATT CCGTTCGATT CCATTCCATT CGATTGCATT 1450

CCATTCGAAT CCTTTCCATT CCATTTCATT CCATTCCTTT CTATTCCATT 1500 CCATTTCATT CGATTTGATT CCATTCTGTT CTATTCCATT CAATTCTTTT 1550

TCATTCCATT CGAATCCTTT CTATTGCAGT CCATTCCATT CGAGTCCATT 1600

CCAATCCCTT CCATTCCATT CCATTACAGT CCATTCCAAT AGATTCCATT 1650 CCTTTGCCTT CCATTCGAAT CCATTCCATT CTAGTCCATT CCATTTGAGT 1700

CAATTCCATT CCATTCCATT CTATTCCTTT CCAATCCATT CGATTCCATT 1750

CGATTCAATT CCATTTGATT CTCTTTCATT CTATTTTATT CCATGCCATT 1800

TTATTGCGTT GCATTCCATT CCGTTTGATT CCAGTCCATT CAAGAAAGTT 1850 CCATTCCAGT CCATTGCTTT CCAGTCCATT CCATTCCACT CTTGTCTATT 1900

CCACTCCATT CCTTTCCATT CCATTCCATA CTATTCCATT CCATTCCTTT 1950

GCATTCCGTT TCCAATCTAT TCGAGTCCAT TGCATTCCAG TCCAATCCAT 2000

TCCATTACAT TCCTTTTGGT TCCCTGCCAG TCGATTGCAT TGCATACTAG 2050

ACCATTCCAA ACCAGTCCAT TCCATTCTAT TTCAACACTT TCCATTCCAC 2100 TCTGTTCGAG TCCATTCCAT TCCAGTCCAT TTAATTCAAG GGCATTCCAT 2150

TCCATTCCAG TCCATTTCAT GTTATTCCAT TCCATTCAAT TCCATTCCAG 2200

ATGATTCCAT TCCATTCTAT ACCATTGCTC TCTGTTCCAT TCCATTCCAT 2250

CTGTCTCCAT TCCTTTCGTT TCGATTCCTT TCCATTCCAT TCCATTACAT 2300

TTGATCCTAT TTTATTAAAT TGCATTCTAT TCGAGTGATT TCCCTTCGAG 2350 TCCTTTCCAT TCAATTCCAT TCCATTCTAT TCCATTCCTT TGGATTCCAT 2400

TCCATTCCGT TCCGTTCACA TCAATTCCTT GTGATTCCAT TACATTCGAT 2450

TTCTTGCCAT TCGATTCCAT TCCTTTTGAC TCCATTTCAT TCGATTCCAT 2500

TCCATTCCAT TAATTTCCAT TCCATTCGAG ACCTTTCCAT TGCAGTCTTT 2550

TCCCTTCGAG TCCATTCCGT TCGATTCCCT TCCATTCGAT TCCATTCCAT 2600 TGGAGTCCGT ACCAGTCGAG TCCATTCTAT TCCAGTCCAT TAGTTTCGAC 2650

TCCATTGCAT TCGAGTGCAT TCCATTCCGT GGTTGTCCAT TCCATTCCGT 2700

TTGATGCCAT TCCATACGAT TCCATTCAAT TCGAGACCAT TCTATTCCTG 2750

TCCATTCCTT GTGGTTCGAT TCCATTTCAC TCTAGTCCAT TCCATTCCAT 2800

TCAATTCCAT TCGACTCTAT TCCGTTCCAT TCAATTCCAT TCCATTCGAT 2950 TCCATTTTTT TCGAGAACCT TCCATTACAC TCCCTTCCAT TCCAGTGCAT 3000

TCCATTCCAG TCTCTTCAGT TCGATTCCAT TCCATTCGTT TCGATTCCTT 3050

TCCATTCCAG CCCATTCCAT TCCATTCCAT TCCTTTCCTT TCCGTTTCAT 3100

TAGATTCCAT TGCATTCGAT TCCATTCAAT TCAATTCCGT GCTATTCAAT 3150

TTGATTCATT TCCATTTAAT TCCATTCCAT TAGATTCCAT TCCGTACGAT 3200 TCCATTCCTT TTGAATCCAT TCCATTGGAG TCCATTCACT TCCAGAACAT 3250

TCCATTCCAG TCGAATCCAT TCGAGTACAT ACCATTAAAG TTCATTACAT 3300

TCTAATACAT TCCATTCCAT TGCATTCCAT TCCATTCCAT TAGATGCCAT 3350

TCGATTCCAT TCCATGCCAA ATCATTGCAT TCCTTTCCAT TCCGTTCCTA 3400

TCAATTCTAT TCCATTCGAT TTAGTTCGAT TCTATTCACT TCCATTCCAT 3450 TCGATTCCAG TCCATTGGAG TCAATTCCTT TCGACACCCA GCCTTTCCAG 3500

TCAATGATTT TGGATTCCAT TTTTTTGCAT TCCATTACAT TCTATGACAT 3550

TCGATTCCGT TTCATTGCAT TCCATTCCAT ACATTTTTAT TCCATTCGAG 3600 ACCGTAGCAT TCCACTTTAT TCCAGGCCTG TCCATTACAC TACATTCCCT 3650

TCCATTCCAA TGAA 3664

"Human alphoid repetitive DNA LI.84 mapping to chromosome 18", Gen Bank

Accession Number X03693, See P. Devilee et al, Nucleic Acids Res., vol. 14, pp 2059- 2073 (1986), a 684 base pair repeat had the following sequence:

AATTCATCAA ATTGCAGACT GCAGCGTTCA GACTGCAGCG TTCTGAGAAA 50

CATCTTTGTG ATGTTTGTAT TCAGGACACC AGAGTTGAAC ATTCCCTATC 100

ATAGAGCAGG TTTGAATCAC TCCTTTTGTA GTATCTGGAA GTGGACATTT 150

GGAGGCTTTC AGGCCTATGT TGGAAAAGGA AATATCTTCC ATAACAACTA 200

GACAGAAGCA TTCTCAGAAC TTATTTGAGA TGTGTGTACT CACACTAAGA 250 GAATTGAACC ACCGTTTTGA AGGAGCAGTT TTGAAACACT CTTTTTCTGG 300

AATCTGCAAA GTGGATATTT GGCTAGCTTT GGGGATTTCG CTGGAACGGA 350

ATACATATAA AAAGCACACA GCAGCGTTCT GAGAAACTGC TTTCTGATGT 400

TTGCATTCAA GTCAAAAGTT GAACACTCCC TTTCATAGAG CAGTCCTGAA 450

ACACTCTTTT GTAGTATCTG GAACTGGACT TTTGGAGCGC TTTCAGGGCT 500 AAGGTGAAAA AGAAATATCT TCCCATAAAA ACTGGACAGA ATCATTCTCA 550

GAAACTTGTT TATGCTGTAT CTACTCAACT AACAAAGTTG AACCTTTCTT 600

TTGATAGAGC AGTTTTGAAA TGCTCTTTTT GTGGAATCTG CAAGTGGATA 650

TTTGGTTAGT TTTGAGGATT TCGTTGGAAG CGGG 684

"Human satellite III DNA fragment, 5' end" Gen Bank Accession number M25431, See H.J. Cooke and J. Hindley, Nucleic Acid Res., vol. 6, 3177-3197 ( 1979), had the following sequence:

AATTCATTTG AAGACAATTC CATTCAATAC CAATTGATGA TGGTTATTTT 50 TGATTCCATT TGATGATGAT TACATTCCAT TTCATCATAA TTCCATTCGA 100 TTCCACTCGA GATTCCATTC GATTCCATTC AA 132

"Human alphoid repetitive DNA LI.26 mapping to chromosome 13 and 21", Gen Bank Accession number X03692, See P. Devilee et al., Nucleic Acid Res.. vol. 14, 2059-2073 (1986), had the following sequence:

AATTCAAATA AAAGGTAGAC AGCAGCATTC TCAGAAATTG CTTTCTGATG 50

TCTGCATTCA ACTCATAGAG TTGAAGATTC CCTTTCATAG AGCAGGTTTG 100

AAACACTCTT TCTGGAGTAT CTGGATGTGG ACATTTGGAG CGCTTTGATG 150

CCTACGGTGG AAAAGTAAAT ATCTTCCCAT AAAAACGAGA CAGAAGGATT 200 CTCAGAAACA AGTTTGTGAT GTGTGTACTC AGCTAACAGA GTGGAACCTT 250

TCTTTTTACA GAGCAGCTTT GAAACTCTAT TTTTGTGGAT TCTGCAAATT 300

GATATTTAGA TTGCTTTAAC GATATCGTTG GAAAAGGGAA TATCGTCATA 350

CAAAATCTAG ACAGAAGCAT TCTCACAAAC TTCTTTGTGA TGTGTGTCCT 400

CAACTAACAG AGTTGAACCT TTCTTTTGAT GCAGCAATTT GGAAACACCT 450 TTTGGTAGAA AATGTAAGTG GATATTTGGA TAGCTTAACG ATTTCGTTGG 500

AAACGGGAAT ATCATCATCT AAAATCTAGA CAGAAGCACT ATTAAGAAAC 550

TACTTGGTGA TATCTGCATT CAAGTCACAG AGTTGAACAT TCCCTTACTT 600

TGAGCACGTT TGAAACACTC TTTTGGAAGA ATCTGGAAGT GGACATTTGG 650

AGCGCTTTGA TGCCTTTGGT GAAAAGGAAA CGTCTTCCAA TAAAAGCCAG 700 ACAGAAGCAT TCTCAGAAAC TTGTTCGTGA TGTGTGTACT CAACTAAAAG 750

AGTTGAACCT TTCTATTGAT AGAGCAGTTT TGAAACACTC TTTTTGTGGA 800

TTCTGCAAGT GGATATTTGG ATTGCTTTGA GGATTTCGTT GGAAGCGGG 849

PREFERRED PROBES FOR CHROMOSOME Y

Oligo # Sequence

6 GAGCCCTTTC AATTTGAGTC CATTC

7 GAGTCGATTT TATTGCATTA GATTC 9 AGAGGACATT CCATTCCAAT GCATT

1 1 TTCTATTCCC TTCTACTGCA TACAA

16 TTGGATTACT TTCCATTCGA TTACA

25 TCTATTCTAT TACATAACTT TCCAT

43 ACAGTCCATT CCAATAGATT CCATT 54 ACTAGACCAT TCCAAACCAG TCCAT

PREFERRED PROBES FOR CHROMOSOME X

Oligo # Sequence

1 TCGAAACGGG TATATGCTCA CGTAA

8 ATCGGGAATA TTTCCACAGA AAAAC

15 GAAGTTTCCC TTTGATAGCG CAGTT

16 TGACACACTT TTTCTACAAT GTGCA 68 ACTAAACACA AACAATCTGA GAAAG

70 CGCAGAGATG AACCTGCCTT TGAGA

75 TGAAATATTC TTTTGGCAGA ATCTG

79 GACGAGAGAG AAGCATTGTC AGAAA

PREFERRED PROBES FOR CHROMOSOME 18

Oligo # Sequence

10 TCTTAGTGTG AGTACACACA TCTCA 10OR CTCACACTAA GAGAATTGAA CCACC

PREFERRED PROBES FOR CHROMOSOME 1

Probe # Sequence

T1A-02 ATCATCATCA AATGGAATCA AAAATA

Tl-01 AATTCATTTG AAGACAATTC CATTCA

PREFERRED PROBES FOR SIMULTANEOUS DETECTION OF CHROMOSOMES 13 AND 21

Probe # Sequence

1 AATTCAAATA AAAGGTAGAC AGCAG

8 CCCATAAAAA CGAGACAGAA GGATT

13 GATATTTAGA TTGCTTTAAC GATAT

MAP POSITIONS OF X CHROMOSOME NONPREFERRED PROBES

The following are the map positions of the nucleotides at the 5' end of various nonpreferred X chromosome probes, each 25 nucleotides in length. The map used is that for the "Human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat".

Probe # 5' position

18 201

19 226

33 576

62 1276

41 776

42 801

43 826

44 851

45 876

46 901

47 926

60 1226

61 1251

65 1351

66 1376

12 51

17 176

21 276

26 401

29 476

74 1826

35 626 35

MAP POSITIONS OF Y CHROMOSOME NONPREFERRED PROBES

The following are the map positions of the nucleotides at the 5' end of various nonpreferred Y chromosome probes, each 25 nucleotides in length. The map used is that for the "Human Y-Chromosome specific repetitive DNA Family (DYZl) Sequence"

Probe # 5' position

8 574

10 801

13 126

17 501

18 526

19 601

20 716

22 736

23 881

26 986

28 1056

47 1761

MAP POSITIONS OF NONPREFERRED PROBES FOR CHROMOSOME 1

The following are the map positions of the nucleotides at the 5' end of various nonpreferred chromosome 1 probes, each 25 nucleotides in length. The map used is that for the "Human satellite III DNA fragment, 5' end", unless the map position is marked with an asterisk (*). If the map position is marked with an asterisk, that position denotes the 3' position of a sequence complementary to the probe. For example, for probe T1A-2, the 3' end of its sequence is a "T" complementary to the "A" at position 46 on the map, the 5' end of its sequence is an "A" complementary to the "T" at position 71 on the map.

Probe # map position (5' if not marked with *)

Tl-1 1

Tl-3 61

Tl-4 91

T1A-2 46*

T1A-3 76*

T1A-4 106*

MAP POSITIONS OF NONPREFERRED PROBES FOR CHROMOSOME 18

The following are the map positions of the nucleotides at the 5' end of various nonpreferred chromosome 18 probes, each 25 nucleotides in length. The map used is that for the "Human alphoid repetitive DNA LI.84 mapping to chromosome 18", unless the map position is marked with an asterisk (*). If the map position is marked with an asterisk, that position denotes the 3' position of a sequence complementary to the probe.

Probe # map position (5' if not marked with *) 10-OL 213

1 1 251 *

11 -OR 264

1 1

3 51 ^' 12 276

14 326

MAP POSITIONS OF NONPREFERRED PROBES FOR CHROMOSOME 13/21

The following are the map positions of the nucleotides at the 5' end of various nonpreferred chromosome 13/21 probes, each 25 nucleotides in length. The map used is that for "Human alphoid repetitive DNA L1.26 mapping to chromosome 13 and 21".

Probe # map position

3 51

5 101

10 226

18 426 23 551

25 626 38 EXAMPLES

Example 1 Pools of X Chromosome Probes

Oligonucleotides, 25-nucleotides long, were made with sequences found in the "human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat". The dye on the 5' end of the Aminolink 2 linker of each oligonucleotide was tetramethylrhodamine. The cells were female white blood cells (WBC).

A good hybridization result was considered to be one in which two tight dots were observed.

Three oligonucleotide pools were made: Pool 1 had equal amounts of oligonucleotides 1, 8, 15, and 16. Pool 2 had equal amounts of oligonucleotides 18, 19, 33 and 62.

Pool 3 had equal amounts of oligonucleotides 68, 70, 75 and 79.

In each pool, the concentration of each oligonucleotide was 10 ug/ml. Hybridization was carried out for 15 min at 85 °C. When pools 1 and 2 were both present in the hybridization cocktail, there were some cells ( about 70 % of the cells) with both one tight dot and one split dot. There were also cells (about 30 % of the cells) with a diffuse signal.

When pools 2 and 3 were present in the hybridization reaction, there were some cells split dots (about 80% of the cells) and a few (about 10 % of the cells) with a diffuse signal. The remaining 10% of the cells had two tight dots.

When pools 1 and 3 were included in the hybridization reaction there were a few cells with split dots (about 10% of the cells) and no cells with diffuse signals. The remaining 90% of the cells had two tight dots.

It was concluded that the combination of Pools 1 and 3 was a high quality combination and gave better results than either the combination of Pools 1 and 2 or the combination of Pools 2 and 3. Example 2 Individual X Chromosome Probes

Oligonucleotides, 25-nucleotides long, were made with sequences found in the "human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat". The dye on the 5' end of the Aminolink 2 linker of each oligonucleotide was tetramethylrhodamine.

The concentration of individual probes in the hybridization assay was 50 ug/ml. The cells were SiHA Cells (Female, polyploid cells with 4 to 8 X-chromosomes). A good result ("specific") was one in which 4-8 tight dots were observed.

A poor result was one in which there was a "nonspecific" result (one in which there was signal from more than eight tight dots or in which there was no signal observed under the fluorescent microscope. The results were as follows:

Probe No. Result

8 specific

41 nonspecific

42 nonspecific

43 nonspecific

44 nonspecific

45 specific

46 nonspecific

47 uncertain

60 No signal

61 uncertain

62 Specific - 4 or 8 dots

65 uncertain

66 nonspecific

70 Maybe

12 no signal

16 specific (weak)

17 No signal

18 Uncertain

21 Specific (weak)

26 specific (weak)

29 no signal

33 specific

74 no signal

75 uncertain

79 uncertain

35 nonspecific This was one of several experiments done to test probe specificity. On the basis of this experiment, probes that either gave no signal or were nonspecific could be excluded from further consideration as high quality, preferred probes. In at least one case (probe 44) specific results were obtained in another experiment, but the lack of consistent specific results caused it to be less than preferred. Those characterized as "specific" or "result uncertain" were still considered to be preferred probes, unless shown to be otherwise by the results of other experiments.

. Example 3

Pool of Y chromosome probes

One probe pool was tested. The pool contained equal concentrations of probe numbers 6, 7, 9, 1 1, 16, 25, 43, 54 each at a concentration of 5 ug/ml. The probes were each 25 nucleotides long and had a nucleotide sequence contained in the Y

Chromosome Centromere, the "Human Y-Chromosome specific repetitive DNA Family

(DYZl) Sequence".

The dye at the 5' end of each probe was tetramethylrhodamine.

Hybridizations were done to "XX + 18" cells (female cells with chromosome 18 trisomy; cells and instructions for maintaining them in culture were obtained from Coriell Institute for Medical Research, Camden, NJ, its catalogue No. GM 03538) and "XY + 21 " cells

(male cells with a chromosome 21 trisomy; cells and instructions for maintaining them in culture were obtained from Coriell Institute for Medical Research, Camden, NJ, its catalogue No. GM 04592A). The criterion for a good result was: one tight dot when XY + 21 cells were used and no signal when XX + 21 cells were used.

The results with "XY + 21" cells were: one bright, tight dot. The results with "XX + 18 cells" were: no signal or dot. Example 4 Individual Y Chromosome Centromere Probes

The cells used were "XX + 18" cells.

The dye at the 5' end of each probe was tetramethylrhodamine. The criterion for a good result was: No signal. The results were:

Probe Result

2 Very faint; Non-specific

6 Good; Very specific

7 Good; Very specific

8 Very faint; Non-specific

9 Good; Very specific

10 Good

11 Good; Very specific

13 Good

16 Good; Very specific

17 Non-specific

18 Non-specific

19 Good

20 Strong; Non-specific

22 Non-specific

23 Non-specific

25 Good; Specific

26 Non-specific

28 Non-specific

43 Good; Specific

47 Good

54 Good; Specific

This was one of several experiments done to test probe specificity. On the basis of this experiment, probes that were nonspecific could be excluded from further consideration as high quality, preferred probes. In some cases, specific results were obtained in another experiment, but the lack of consistent specific results caused it to be less than preferred. Those characterized as "good" were considered to be candidates to be preferred probes, unless shown otherwise by the results of other experiments. Example 5

Chromosome 18 Probe Populations

1) Five oligonucleotides were tested individually (not as part of a pool), the oligonucleotides were numbers 10, 10-OL, 10-OR, 1 1 , and l l-OR. Oligonucleotides 10 and 1 1 were each 25-nucleotides long had each had a nucleotide sequence present in the centromeric DNA strand complementary to that specified herein as "Human alphoid repetitive DNA L1.84 mapping lo chromosome 18". Oligonucleotides 10-OL and 10-OR base sequences were partially complementary to that of oligonucleotide 10, and were targeted for the centromere strand specified herein as "Human alphoid repetitive DNA LI.84 mapping to chromosome 18. Oligonucleotide 1 1-OR was partially complementary to probe 1 1 and was targeted for the centromere strand specified herein as "Human alphoid repetitive DNA LI.84 mapping to chromosome 18.

The dye attached at the 5' end of each probe was tetramethylrhodamine. Cells were "XX + 18" cells.

The concentration of each probe in the hybridization cocktail was 80 ug/ml. The criterion for a good result was: three tight dots. The results were as follows:

Probe 10: The amount of signal other than three tight dots, ranged from none to very little.

Probe 10-OL: In addition to three tight dots, nonspecific signal was observed. Probe 10-OR: Three tight dots, no non-specific signal. Probe 11 : In addition to three tight dots, there was strong non-specific signal.

Probe 11-OR: In addition to .three tight dots, there was a very strong non-specific signal.

As a result it was concluded that probes 10 and 10-OR are high quality, preferred probes. Example 6 Probes for Chromosome 18

1) Six oligonucleotides were tested individually (not as part of a pool), the oligonucleotides were numbers 1, 3, 10, 11, 12, and 14. The oligonucleotides, each 25- nuclcotidcs long, each had a nucleotide sequence complementary to one shown herein for "Human alphoid repetitive DNA LI.84 mapping to chromosome 18". The dye attached at the 5' end of each probe was tetramethylrhodamine.

Cells were female (XX) human white blood cells The criterion for a good result was: two tight dots Results were as follows: Probe No. Results

1 2 dots diffuse, some non-specific signal

3 2 dots (diffuse)

10 2 tight dots-looks good

11 2 tight dots-looks good

12 nonspecific - 4 dots

14 nonspecific - 4 dots

This was one of several experiments done to test probe specificity. On the basis of this experiment, probes that were nonspecific could be excluded from further consideration as high quality, preferred probes. Those that were specific were still considered to be high quality, preferred probes, unless shown to be otherwise by the results of other experiments. Example 7 Probes for Chromosome number 1 The cells used were human white blood cells (WBC) with two "number 1 " chromosomes. The target for hybridization was either WBC or a chromosome spread from WBC.

The dye at the 5' end of each probe was tetramethylrhodamine. The criterion for a good result was: two tight dots The results were:

Probe Target Result

T 1 - 1 spread 1 bright dot plus signal from a chromosome other than chromosome 1 Tl WBC 1 bright dot plus signal from a chromosome other than chromosome 1

Tl-2 WBC 2 bright dots Tl-2 spread 2 bright dots plus 2 faint dots

T1A-1 WBC 2 dots, very bright T1A-1 spread 2 dots, very bright

T1A-3 WBC 2 tight dots plus 3 nonspecific dots T1A-3 spread 2 tight dots plus 3 nonspecific dots

T1A-4 spread signal from four chromosomes

This was one of several experiments done to test probe specificity. On the basis of this experiment, probes that were nonspecific could be excluded from further consideration as high quality, preferred probes. On the basis of this experiment, probes Tl-2 and T1A-

1 were considered to be preferred high quality probes. This was true even though Tl-2, in addition to generating the desired two specific dots also generated two faint dots. Example 8 Centromere Probes for Chromosome 13 and 21

The centromere sequences from chromosomes 13 and 21 are identical. Therefore, the probes should hybridize equally to chromosome 13 and chromosome 21.

The cells used were human cell line with trisomy for chromosome 21 (GM 02419, XX+21) obtained from Coriell Institute for Medical Research.

The criterion for a good result was: 5 dots, 3 for chromosome 21 (trisomy 21) and 2 for chromosome 13.

Probe Result

1 Very specific, 5 dots

3 Non-specific, more than 5 dots 5 Non-specific, more than 5 dots

8 Very specific, 5 dots

10 Non-specific, more than 5 dots

13 Very specific, 5 dots

18 Non-specific, more than 5 dots 23 Specific, 5 dots plus some faint non¬ specific

26 Non-specific, more than 5 dots

Specific probes 1, 8, 13, and 23 were pooled in different combinations and were hybridized to trisomy 21 cell lines (XX + 21), the results are described below:

Probe Combination Result

#23, 1, 8 Non-specific

#23, 1, 3 5 dots plus non-specific

#23, 8, 13 5 dots plus non-specific #1, 8, 13 Specific 5 dots, no non-specific

The probe combination 1 , 8 and 13 gave the specific 5 dots without any non-specific dots or signal. Therefore, probes 1, 8 and 13 are highly specific probes. Example 9 Analysis of Probes by Southern Blot Hybridization

Southern Blot hybridization was performed to identify probes that were non- specific. A probe was considered nonspecific if it hybridized to more than one band on a

Southern Blot. The procedure can be summarized as follows:

Cellular DNA was purified and digested to completion with restriction enzyme Eco-Rl. It was then fractionated by electrophoresis and transferred in denatured form to, and immobilized on, nitrocellulose paper. Probes labelled with ³²P were hybridized against the immobilized restriction enzyme DNA fragments in sodium phosphate buffer + 0.1% SDS at 50 °C for 12 hours. The filter paper was then washed in and analyzed for bound probe by.

For the X chromosome, 60 non-overlapping probes from the Human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat region were tested. Of these, 33 were rejected as being nonspecific.

For the Y chromosome, 52 non-overlapping probes from the "Human Y- Chromosome specific repetitive DNA Family (DYZl) Sequence centromere region were tested. Of these, 18 were rejected as being nonspecific.

For the chromosome 18, 30 non-overlapping probes mapping to the Human alphoid repetitive DNA LI.84 centromere region were tested. Of these, 18 were rejected as being nonspecific.

Claims

47CLAIMSWHAT IS CLAIMED IS:

1. An oligonucleotide probe population specific for a target that is the human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat, said probe population comprising at least three different probes, each having a probe nucleotide sequence different from the nucleotide sequences of the other two probes and not complementary to the nucleotide sequences of the other two probes, each of said three probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, such that each of said three probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of sixteen probe nucleotide sequences,

TCGAAACGGG TATATGCTCA CGTAA and its complement, ATCGGGAATA TTTCCACAGA AAAAC and its complement, GAAGTTTCCC TTTGATAGCG CAGTT and its complement,

TGACACACTT TTTCTACAAT GTGCA and its complement, ACTAAACACA AACAATCTGA GAAAG and its complement, CGCAGAGATG AACCTGCCTT TGAGA and its complement, TGAAATATTC TTTTGGCAGA ATCTG and its complement, GACGAGAGAG AAGCATTGTC AGAAA and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said three probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing sixteen probe nucleotide sequences.

2. An oligonucleotide probe population of Claim 1 , said probe population comprising at least eight different probes, each having a probe nucleotide sequence different from that of the other seven probes and not complementary to any of them, each of said eight probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, each of said probe nucleotide sequences that is 25 or more nucleotides being selected from the group of sixteen probe nucleotide sequences specified in Claim 1 , each of said probe nucleotide sequences that is less than 25 nucleotides being entirely from part of one of said group of sixteen probe nucleotide sequences.

3. A probe population of Claim 1 wherein probe nucleotide sequences selected from the group of 16 sequences account for all of the sequences that hybridize to the target that is the human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat.

4. A probe population of Claim 2 wherein probe nucleotide sequences selected from the group of 16 sequences account for all of the sequences that hybridize to the target that is the human chromosome X-linked alpha satellite repetitive DNA 2.0 kb repeat.

5. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence ATCGGGAATA TTTCCACAGA AAAAC or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

6. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence GAAGTTTCCC TTTGATAGCG CAGTT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

7. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence TGACACACTT TTTCTACAAT GTGCA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

8. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence ACTAAACACA AACAATCTGA GAAAG or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

9. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence CGCAGΛGATG ΛACCTGCCTT TGAGA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

10. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence TGAAATATTC TTTTGGCAGA ATCTG or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

1 1. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence GACGAGAGAG AAGCATTGTC AGAAA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

12. An oligonucleotide probe population specific for a target that is the human Y- chromosome specific repetitive DNA Family (DYZl) sequence, said probe population comprising at least three different probes, each having a probe nucleotide sequence different from the nucleotide sequences of the other two probes and not complementary to the nucleotide sequences of the other two probes, each of said three probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, such that each of said three probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of sixteen probe nucleotide sequences,

GAGCCCTTTC AATTTGAGTC CATTC and its complement, GAGTCGATTT TATTGCATTA GATTC and its complement, AGAGGACATT CCATTCCAAT GCATT and its complement, TTCTATTCCC TTCTACTGCA TACAA and its complement, TTGGATTACT TTCCATTCGA TTACA and its complement, TCTATTCTAT TACATAACTT TCCAT and its complement, ACAGTCCATT CCAATAGATT CCATT and its complement,

ACTAGACCAT TCCAAACCAG TCCAT and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said three probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing sixteen probe nucleotide sequences.

13. An oligonucleotide probe population of Claim 12, said probe population comprising at least eight different probes, each having a probe nucleotide sequence different from that of the other seven probes and not complementary to any of them, each of said eight probe nucleotide sequences complementary to a sequence of 20 to 30 nucleotides in said target, each of said probe nucleotide sequences that is 25 or more nucleotides being selected from the group of sixteen probe nucleotide sequences specified in Claim 12, each of said probe nucleotide sequences that is less than 25 nucleotides being entirely from part of one of said group of sixteen probe nucleotide sequences.

14. A probe population of Claim 12 wherein probe nucleotide sequences selected from the group of 16 sequences account for all of the sequences that hybridize to the target that is the human Y-chromosome specific repetitive DNA Family (DYZl) sequence.

15. A probe population of Claim 13 wherein probe nucleotide sequences selected from the group of 16 sequences account for all of the sequences that hybridize to the target that is the human Y-chromosome specific repetitive DNA Family (DYZl) sequence.

16. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence GAGCCCTTTC AATTTGAGTC CATTC or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

17. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence GAGTCGATTT TATTGCATTA GATTC or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

18. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence AGAGGACATT CCATTCCAAT GCATT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

19. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence TrCTATTCCC TTCTACTGCA TACAA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

20. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence TTGGATTACT TTCCATTCGA TTACA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

21. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence TCTATTCTAT TACATAACTT TCCAT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

22. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence ACAGTCCATT CCAATAGATT CCATT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

23. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence ACTAGACCAT TCCAAACCAG TCCAT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

24. An oligonucleotide probe population specific for a target that is the human alphoid repetitive DNA LI.84 mapping to chromosome 18, said probe population comprising at least two different probes whose nucleotide sequences are partially complementary to each other, each having a probe nucleotide sequence different from the nucleotide sequence of the other and not completely complementary to the nucleotide sequence of the other, each of said two probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, such that each of said two probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of four nucleotide sequences,

TCTTAGTGTG AGTACACACA TCTCA and its complement,

CTCACACTAA GAGAATTGAA CCACC and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said probe nucleotide sequences that is less than 25 nucleotides being entirely from part of one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing four probe nucleotide sequences.

25. A probe population of Claim 24 wherein probes with nucleotide sequences selected from the group of four sequences account for all of the sequences that hybridize to the target that is human alphoid repetitive DNA LI.84 mapping to chromosome 18.

26. A probe of not more than 40 nucleotides that comprises a sequence of at least twenty nucleotides from either the sequence TCTTAGTGTG AGTACACACA TCTCA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

27. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence CTCACACTAA GAGAATTGAA CCACC or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

28. An oligonucleotide probe population specific for a target that is the human chromosome 1 III DNA fragment, 5' end, 132 BP repeat, said probe population comprising at least two different probes, each having a probe nucleotide sequence different from the nucleotide sequence of the other and not completely complementary to the nucleotide sequence of the other probe, each of said two probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, such that each of said two probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of four nucleotide sequences,

ATCATCATCA AATGGAATCA AAAATA and its complement, AATTCATTTG AAGACAATTC CATTCA and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing sixteen probe nucleotide sequences, such that, in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing four probe nucleotide sequences.

29. A probe population of Claim 28 wherein probes with nucleotide sequences selected from the group of four sequences account for all of the sequences that hybridize to the target is the human chromosome 1 III DNA fragment, 5' end, 132 BP repeat.

30. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides either the sequence ATCATCATCA AATGGAATCA AAAATA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

31. A probe of not more than 40 nucleotides that comprises a sequence of at least

20 nucleotides from either the sequence AATTCATTTG AAGACAATTC CATTCA or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

32. A process of detecting a nucleic acid target molecule in a human cell or chromosome spread, which process comprises the steps of:

(1) co-incubating the human cell or chromosome spread with an oligonucleotide probe population of Claim 1,

(2) detecting the oligonucleotide probe molecules that are hybridized to the target molecule; such that step (1) is performed under conditions that allow the oligonucleotide probe to hybridize to the target molecule.

33. A process of Claim 32 wherein the nucleic acid target molecule is in a human cell and step (1) comprises co-incubating the human cell with an oligonucleotide population of Claim 1.

34. A process of detecting a nucleic acid target molecule in a human cell or chromosome spread, which process comprises the steps of:

( 1 ) co-incubating the human cell or chromosome spread with an oligonucleotide probe population of Claim 12, (2) detecting the oligonucleotide probe molecules that are hybridized to the target molecule; such that step (1) is performed under conditions that allow the oligonucleotide probe to hybridize to the target molecule.

35. A process of Claim 34 wherein the nucleic acid target molecule is in a human cell and step (1) comprises co-incubating the human cell with an oligonucleotide population of Claim 1.

36. A process of detecting a nucleic acid target molecule in a human cell or chromosome spread, which process comprises the steps of:

( 1) co-incubating the cell or chromosome spread with an oligonucleotide probe population of Claim 24,

(2) detecting the oligonucleotide probe molecules that are hybridized to the target molecule; such that step (1) is performed under conditions that allow the oligonucleotide probe to hybridize to the target molecule.

37. A process of Claim 36 wherein the nucleic acid target molecule is in a human cell and step (1) comprises co-incubating the human cell with an oligonucleotide population of Claim 1.

38. A process of detecting a nucleic acid target molecule in a human cell or chromosome spread, which process comprises the steps of:

(1) co-incubating the cell or chromosome spread with an oligonucleotide probe population of Claim 28,

(2) detecting the oligonucleotide probe molecules that are hybridized to the target molecule; such that step (1) is performed under conditions that allow the oligonucleotide probe to hybridize to the target molecule.

39. A process of Claim 38 wherein the nucleic acid target molecule is in a human cell and step (1) comprises co-incubating the human cell with an oligonucleotide population of Claim 1.

40. An oligonucleotide probe population specific for a target that is human alphoid repetitive DNA LI.26 mapping to chromosomes 13 and 21 , said probe population comprising at least three different probes, each having a probe nucleotide sequence different from the nucleotide sequence of the other two probes and not completely complementary to the nucleotide sequence of the other two probes, each of said three probe nucleotide sequences complementary to a sequence of 13 to 30 nucleotides in said target, such that each of said probe nucleotide sequences that is 25 or more nucleotides comprises a sequence selected from the following group of six nucleotide sequences, AATTCAAATA AAAGGTAGAC AGCAG and its complement, CCCATAAAAA CGAGACAGAA GGATT and its complement, GATATTTAGA TTGCTTTAAC GAT AT and its complement, when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA, each of said probe nucleotide sequences that is less than 25 nucleotides being the same as part of one of the foregoing six probe nucleotide sequences, such that in the probe population, the sum of the lengths of the probe nucleotide sequences is not more than twice the sum contributed by the foregoing six probe sequences.

41. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence AATTCAAATA AAAGGTAGAC AGCAG or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

42. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence

CCCATAAAAA CGAGACAGAA GGATT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

43. A probe of not more than 40 nucleotides that comprises a sequence of at least 20 nucleotides from either the sequence

GATATTTAGA TTGCTTTAAC GAT AT or its complement when a probe is DNA but where the letter T is replaced by the letter U if the probe is RNA.

44. A process of detecting a nucleic acid target molecule in a human cell or chromosome spread, which process comprises the steps of: 1) co-incubating the human cell or chromosome spread with an oligonucleotide probe population of Claim 40,

2) detecting the oligonucleotide probe molecules that are hybridized to the target molecule; such that step (1) is performed under conditions that allow the oligonucleotide probe to hybridize to the target molecule.

45. A kit for analyzing human chromosomes, said kit comprising a probe population of either Claim 1, Claim 12, Claim 24, Claim 28, or Claim 40, and further comprising either instructions for using said probe population to detect a human chromosome or an indication that a use for said probe population is to detect a human chromosome or centromere.