INTRODUCTION The present invention relates generally to the area of organic chemistry, biology, molecular biology, biochemistry and medicine. In particular, it relates to a novel method for detecting a substance through its specific affinity for a molecule which is hybridized to an immobilized chromosome.

BACKGROUND OF THE INVENTION The following description of background of the invention is provided simply as an aid in understanding the present invention and is not admitted to describe or constitute prior art to the present invention.

The pioneering work of A. M. Maxam and W. Gilbert, Proc. Nat'l Acad. Sci. USA, 1977,74: 560- 664 and Sanger, et al., Proc. Nat'l Acad. Sci. USA, 1977,74: 5463-5467 in nucleic acid sequencing ushered in a new era of exploration into the fundamental unit of genetic information in all living organisms, the gene, and into the genetic code, which establishes the correspondence between the sequence of bases in nucleic acids, DNA and RNA, and the sequence of amino acids in proteins.

With the discovery that many diseases and disorders afflicting man involve abnormalities either initially present in the genetic code of an individual (genetically

transferred disorders such as cystic fibrosis, amyotrophic lateral sclerosis (Lou Gehrig's disease), Duchenne muscular dystrophy, etc.) or created in the genetic code by external forces (significantly, cancer), the potential of using the genetic code as a diagnostic tool and a target for therapy became apparent. As a function of this observation, the urgency to define the complete nucleic acid sequence of the human genome (as well as other genetically interesting organisms such as the human gut bacterium, Escherichia coli, baker's yeast, Saccharomyces cerevisiae, the fruit fly, Drosophila melanogaster and the laboratory mouse, Mus musculus) and thereby to gain not only knowledge of the extent of genetic diversity but of the nature of changes therein related to diseases and other disorders, escalated.

Achievement of the above goal, however, was impeded by the fact that the Maxam- Gilbert and Sanger sequencing technologies are linear in nature; i. e., one can examine only one oligonulceotide at a time, and relatively slowly at that due to the use in both procedures of gel electrophoresis to separate either the cleavage (Maxam-Gilbert) or the chain- terminated (Sanger) fragments created by the techniques. While advances in electrophoresis methods, such as ultra-thin gels and capillary arrays, provided incremental improvements, these technologies remained throughput limited by their sequential nature and the speed of resolution of separations.

This state of affairs was dramatically changed by the advent of high-density oligonucleotide probe arrays. In this procedure, large numbers of oligonucleotide fragments such as, for instance, all 1,073,741,824 15-mers, could be created at specific attachment locations on a solid support in such a manner that they would be capable of hybridizing with complementary sequences with which they might come in contact. An unknown oligonucleotide could then be contacted with the array and, by locating the point on the solid support where the unknown oligomer hybridized using visualization techniques such as fluorescent labeling and confocal scanning, one could immediately determine which of the known 15-mers hybridized with the unknown and thereby immediately know the nucleotide sequence of the unknown which would be the complement of the 15-mer with which it hybridized.

To achieve the high-density, information rich format of such conventional high- density probes, however, requires extremely sophisticated technology. For instance, in one procedure (R. J. Lipshutz, et al., Biotechniques, 1995,9 (3): 442-447), high density

oligonucleotide arrays are created using light-directed chemical synthesis which combines semiconductor based photolithography and solid state synthesis. In this process, linkers modified with photochemically removable protecting groups are attached to a solid substrate.

Light is directed through a mask to specific areas of the substrate surface, activating those areas to chemical coupling. The substrate is then contacted with the first of a series of nucleotides, which nucleotides have a photo-labile protection group at the 5'end. Chemical coupling occurs at the sites which have been activated by illumination of the surface through the mask. Next, light is directed through another mask to another portion of the substrate surface, activating it. The chemical coupling cycle is repeated with the second of the series of nucleotides. This process is repeated, using the proper sequence of masks and chemical coupling steps, until a collection of sequence specific oligonucleotides is constructed, each oligonucleotide occupying a different predefined position in the high density array. In this manner, the set of all 15-mers, for instance, can be synthesized in 60 cycles, employing 60 separate masks and 60 separate chemical coupling reactions.

While the above technique is relatively efficient; e. g., the complete set of 15-mers can be created in approximately 10 hours (Lipschutz, et al., ibid.), the sophistication of the procedure for making the arrays; e. g., the creation of the masks, precise registration of the masks for exposure, etc., removes it from the purview of the average investigator who is relegated to reliance on commercially available sets of micro-arrays with which to work.

What is needed is a simple, expedient method of creating high density microarrays in the normal laboratory environment so that a researcher can have the capacity to custom prepare an array to meet the immediate needs of the day. The present invention provides such a method.

SUMMARY OF THE INVENTION In one aspect, the present invention relates to a method for detecting a substance by (1) attaching a chromosome to a solid support; (2) hybridizing a bifunctional molecular probe with the chromosome where the bifunctional molecular probe has at least two parts, or segments, one segment being a polynucleotide in which the sequence of nucleotides is complementary to a sequence of nucleotides at a predefined position in the chromosome, the

second segment being composed of a molecule having a specific affinity for; i. e., the ability to bind to, the substance to be detected; (3) contacting a test solution suspected of containing the substance to be detected with the bifunctional molecular probe; and, (4) examining the chromosome to see if the bifunctional molecular probe hybridized at the pre-selected position of the chromosome has bound any of the substance being tested for.

As used herein, the term"substance"refers to a molecular species that has a specific affinity for another molecule; that is, the substance will be selectively bound by the other molecule when placed in its presence. Examples, without limitation, of substances and molecules which have a specific affinity include, respectively, oligonucleotides, such as DNA and RNA, and their complementary nucleic acid sequences, antigens and their corresponding antibodies, lectins and carbohydrates, enzymes and their corresponding substrates, hormones and their corresponding receptors and genes and the corresponding proteins with which they interact in performing their genetic function.

"Complement"or"complementary"refers to the specific hydrogen bond base pairing of adenine with thymine or uracil (A-T and A-U) and of cytosine with guanine (C-G).

Chains of complementary base pairs having a sufficient number of hydrogen bonds to form a stable two-strand molecule are said to be"hybridized"to one another.

A bifunctional molecular probe refers to a molecule having at least two parts or segments. One segment of the molecule consists of a polynucleotide having a nucleotide sequence that is complementary to the nucleotide sequence at a specific preselected site on a chromosome. The bifunctional molecular probe can, under proper condition, be hybridized with the chromosome at the pre-selected position through the complementary nucleotide sequence. The second segment of the bifunctional molecular probe consists of a molecule for which a target substance of interest has a specific affinity, as described above. It is important to note that the"substance"and the"second segment"of the"bifunctional molecular probe" are interchangeable. That is, the substance to be detected may be a particular enzyme in which case the second segment of the bifunction molecular probe would be a known substrate for that particular enzyme. Conversely, the substance to be detected could be a substance which is a known substrate for a particular enzyme and the second segment of the bifunctional molecular probe could be the enzyme for which the substance is a substrate.

"Detecting"a substance refers to the determination of whether or not any of the substance has in fact bound to a particular bifunctional molecular probe. As used herein, methods of detection include, without limitation, radioisotope labeling, thymidine dimer formation fluorescent labeling, energy transfer fluorescent labeling, chemiluminescent labeling or phosphorescent labeling of the substance prior to exposure to the bifunctional molecular probe and, after exposure of the substance to the bifunctional molecular probe, by examination of the chromosome at the position containing the bifunctional molecular probe with an appropriate device for observing the label; for example, a scintillation counter, a confocal scanner, a light microscope, a spectrophotometer and the like, to determine if the selected label is present.

A"tag"refers to one or more of the above described labeling molecules."Tagging" refers to the procedure of incorporating a label in a substance.

Radioisotope label tagging refers to the incorporation of a radioactive form of an element in a molecule to be incorporated in a substance. Commonly used radioisotopes include, without limitation, 3H, 22Na, 32p, 35S and 125I.

Fluorescent label tagging refers to the incorporation into a substance of a molecule, called a"fluorphor", which absorbs energy at a shorter wavelength and emits energy at a longer wavelength. The emission of energy continues for only so long as the stimulus, i. e., the shorter wavelength radiation, is being applied.

Phosphorescent label tagging refers to the incorporation into a substance of a molecule which absorbs energy at a shorter wavelength and emits energy at a longer wavelength as in fluorescent labeling. The difference between fluorescent labeling and phosphorescent labeling is that a phosphorescent label continues to emit energy after the stimulus has been removed.

An"energy-transfer donor-acceptor pair"refers to two fluorophors one of which absorbs energy at a particular wavelength and then transfers a portion of that energy to the second fluorophor that emits energy at a different wavelength. Energy-transfer donor- acceptor pair label tagging refers to the incorporation into a substance of two such fluorophors at a distance from each another which permits efficient transfer of energy from the donor to the acceptor.

Chemiluminescent label tagging refers to the incorporation into a substance of a molecule that emits visible light as the result of chemical reaction.

Thymidine dimer formation occurs when two adjacent thymidine nucleotides in an oligonucleotide are irradiated with UV light.

As used herein, the term"chromosome"refers to the naturally occurring discrete units in the nucleus of a cell which carry the genetic information of the organism. Chromosomes are composed of DNA, which comprise the genes, and proteins that help to define the structure and the level of activity of the chromosome. In a presently preferred embodiment of this invention, chromosome refers to a salivary gland chromosome of Drosophila melanogaster, the common fruit fly.

A"solid support"refers to an optionally sized piece of flat, rigid or semi-rigid material that may be transparent or opaque and is capable of being treated to immobilize a chromosome. The solid support can be, without limitation, such materials as aluminum, stainless steel, polyethylene, polycarbonate, polyester, glass, silicon and the like. In a presently preferred embodiment of this invention, the solid support comprises a glass microscope slide.

"Attaching"a chromosome to a solid support refers to coating the solid support with a material that is capable of binding a chromosome through ionic and/or hydrogen bonding forces. For example, the solid support can be coated with lysine, which is a positively charged base and which therefore will bind chromosome through ionic interaction. In a presently preferred embodiment of this invention, D. melanogaster chromosome can be attached to a solid support by means of an antibody which is coated on the solid support and which binds the chromosome through both ionic interactions and hydrogen bonding. In a further presently preferred embodiment of this invention, numerous copies of the D. melanogaster chromosome are attached to the solid support.

As used herein,"hybridization"refers to the pairing of complementary nucleic acids of two polynucleotide strands under conditions amenable to formation of stable hydrogen bonds. In the present invention, one polynucleotide strand comprises a portion of a DNA strand at a preselected location of a chromosome attached to the solid support. The other, complementary polynucleotide strand that, under appropriate conditions will hybridize to the chromosome DNA strand, comprises the first segment of the bifunctional molecular probe.

"Contacting"a test solution with the molecule refers to immersing in a solution suspected to contain the target substance, under conditions conducive to binding between the target substance and its specific affinity mate, the solid support to which is attached the chromosome which in turn is hybridized to the molecule which has, as a second segment, the specific affinity mate of the target substance.

"Examining"a chromosome to determine if any of the target substance has adhered to the second segment of the bifunctional molecular probe which is hybridized at a pre-selected location on chromosome refers to employing the techniques described under the above definition of"detecting"to observe whether or not a tag used with a particular target substance is present at the pre-selected location on the chromosome.

In another embodiment of this invention, a method is provided for simultaneously detecting a number of different substances in a test solution. The method comprises (1) attaching a chromosome, preferably many chromosomes, to a solid support; (2) hybridizing a number of different bifunctional molecular probes with the chromosome such that probes having a particular second segment ; i. e., a second segment which is the specific affinity mate of one of the target substances, are hybridized through their first segments at a predetermined specific location within the chromosome by virtue of the first segment being a polynucleotide having a nucleotide sequence which is uniquely complementary to the nucleotide sequence at the predetermined location on chromosome; (3) contacting a test solution suspected to contain one or more of the target substances with the different bifunctional molecular probes bound to the chromosome; and (4) examining the pre-selected locations on the chromosome to which the bifunctional molecular probes had been hybridized to determine if any of the suspected target substances have been bound by the various probes. Since a particular target substance will be bound by a particular probe hybridized at a specific location on the chromosome, the composition of the test solution in terms of the presence of absence of the target substances can be directly read by examination of the preselected locations on the chromosome.

In a further aspect of this invention a device for the simultaneous detection of a plurality of substances is claimed. Such device comprises a solid support to which a plurality of chromosomes have been attached, the chromosomes being also hybridized to a plurality of different bifunctional molecular probes at different pre-selected locations on the

chromosomes. The bifunctional molecular probes hybridized at each pre-selected location have a second segment having a specific affinity, and therefore the capability of selectively binding, one of the plurality of substances to be tested for. Contacting a test mixture suspected to contain any of the plurality of substances will result in those substances for which a bifunctional molecular probe has a specific affinity being bound to the device at the pre-selected location on the chromosome where that particular bifunctional molecular probe is hybridized, thus permitting the immediate identification of the substance.

A kit comprising the above device or components thereof together with additional materials for carrying out the methods claimed herein such as, without limitation, fluorophors, radiolabeled nucleotides, phosphorescing agents, means of incorporation tags into substance, PCR reagents, standards for calibrating the method, sets of particular enzymes, antibodies, hormones, antigens, hormone receptors, genes, gene substrates, lectins, etc. to be incorporated into the bifunctional molecular probe, comprise another aspect of this invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a method for detecting substances in a mixture by contacting the mixture with a solid support to which has been attached a large number or "array"of chromosomes which, in turn, have been hybridized with a large number or"array" of different bifunctional molecular probes, one segment of each different probe being hybridizing with a pre-selected location on the chromosome and a second segment of each different probe at each pre-selected location on said chromosome having a specific affinity, and therefore specific ability to bind, one of the substances suspected to be in the mixture to be tested. The following description describes in detail how the method will function.

This invention introduces the concept of using chromosomes from eucaryotic cell as DNA arrays instead of machine-produced DNA arrays. Eucaryotic cell chromosomes appear during cell division and nuclear DNA is arranged in a linear manner along the length of chromosomes so that a portion of DNA with a specific nucleotide sequence is situated at a specific site on the chromosomes. Furthermore, chromosomes are banded at fixed points along their lengths and a specific nucleotide sequence is situated at a morphologically

definable position relative to the bands. Therefore, eucaryotic cell chromosomes are natural DNA arrays.

The number of chromosomes varies from specie to specie and the length of chromosomes varies from cell to cell. For example, a human somatic cell contains 23 pairs of chromosomes; the length of the largest metaphase chromosome is about 10 micrometers and the length of the smallest is about 1 micrometer in a small lymphocyte. Drosophila melanogaster salivary gland cell contains four giant polytene chromosomes; the length of the largest chromosome is about 850 micrometers and the length of the smallest is about 30 micrometers. In both species, the DNA libraries are constructed from segmented nuclear DNA. From the libraries, vast numbers of recombinant plasmid clones are made and, to date, several thousand of the clones of the DNA are assigned a specific site on the chromosomes by in situ hybridization. The cloned DNA, with the ability to recognize and to hybridize with a specific site on the chromosomes, may be used to deliver specific materials to specific sites on the chromosomes. This is accomplished by linking the specific material to the cloned DNA and hybridizing it with the chromosomes. The specific materials include reporting markers, known sequences of DNA, known sequences of RNA, proteins (antibodies, receptors, binding proteins, lectins, and others), glycoproteins, carbohydrates, lipids, glycolipids, steroids, drugs, other organic and inorganic compounds, etc.

Drosophila melanogaster salivary gland polytene chromosomes will be used for the DNA array. Drosophila has four pairs of chromosomes: the X/Y sex chromosomes and the autosomes 2,3 and 4. The fourth chromosome is quite small. The size of the genome is about 165 million bases and contains an estimated 12,000 genes. In the salivary glands, each chromosome divides hundreds of times, but all the strands remain attached to each other.

The result is a massively thick polytene chromosome. Polytene chromosomes are easily observed under the microscope and have a pattern of dark and light bands that are unique for each section of the chromosome. As a result, those knowledgeable in the art can recognize a part of the chromosome by reading the polytene bands. For identification of a location on the chromosomes, a standard map of the polytene chromosome is used. The map divides the genome into 102 numbered bands (1-20 is the first, 21-60 is the second, 61-100 the third and 101-102 the fourth); each of the bands is divided into six letter bands (A-F) and each letter band is further subdivided into 1 to 13 numbered divisions. The locations of many genes are

known down to the resolution of a letter band and individual cloned genes can be placed on the polytene map. On the average, a letter band contains about 300kb of DNA and 15-25 genes.

The Drosophila melanogaster genomic P1 libraries were constructed using genomic DNA isolated from a mixture of both male and female animals of the strain iso 1 (y; cn bw sp) by David Smoller (Smoller, D. A., D. Petrov, and D. L. Hartl, Chromosoma, 1991,100: 487-494). High molecular weight DNA extracted from nuclei was partially digested with Sau3A then size fractionated using a 10%-40% sucrose gradient to select fragments in the range of 75 to 100 kb. Recombinant PI clones were generated in two ways using two similar PI cloning vectors, pNS582tetl4AdlO (Sternberg, N., PNAS, 1990,87: 103-107) and pAdlOsacBII (Pierce, J. C., B. Sauer, and N. Sternberg, PNAS, 1992,89: 2056-2060). The pAdlOsacBII contains an insert of a fragment with Bacillus amyloliquifaciens sacB at the BamHI site of the tetracycline resistance gene of pNS582tetl4AdlO. The ligation products of the pNS582tetl4AdlO reaction were packaged into phage heads in vitro and used to infect E. coli strain NS3145 then plated on LB plus kanamycin media to select clones. For the pAdlOsacBII ligation reaction the strain NS3529 was used for infection after in vitro packaging into phage heads. These recombinant clones were selected by plating on LB media containing kanamycin and 5% sucrose.

The genomic PI clones were picked into individual wells of microtiter plates filled with LB media with kanamycin and 10% glycerol. After growth at 37° C overnight the plates were manually replicated using a 96-pin hand tool to provide additional copies of the library.

A total of 40 plates from the pNS583tetl4AdlO ligation and 99 plates from the pAdlOsacBII ligation were picked for a total of 139 plates. An additional 96 microtiter plates from other pAdlOsacBII ligation reactions were later picked. For the pNS582tetl4AdlO clones the average insert size was 83.0 6.2 kb (N=25) and for the pAdlOsacBII clones the average insert size was 82.5 5.8 kb (N=20) (Hartl, D. L., D. I. Nurminskky, R. W. Jones, and E. R.

Lozovskaya, PNAS, 1994,91: 6824-6829).

Each PI clone has a unique identifying number of the form DSnnnnn (where n is a number between 0 and 9). In addition, each PI clone has a position in the arrayed library that is expressed as a microtiter plate number and position number within that plate. This number (Library location) refers to the position of the PI clone in a particular representation of the

library. In each plate the clones in the wells 1 through 40 (A 1 through D4) contain inserts in the PI vector NS582tetl4adlO. In each plate the wells 41-96 (D5 through H12) contain inserts in the PI vector adIOsacBII. Some of the PI clones are completely sequenced and the sequence data are available from the Berkeley Drosophila Genome Project (BDGP).

In one embodiment of this invention, about thirty 30-mer oligonucleotides with an additional two or three adenine-thymidine-thymidine (-ATT) repeats at the 5'and 3'ends will be synthesized. The sequence of each of the 30-mer oligonucleotides will correspond to the DNA clones that have been shown to hybridize to a single site on the chromosomes. The sites will be evenly spaced over three major chromosomes. Prior to the hybridization, the DNA will be exposed to ultraviolet light to convert the thymidine-thymidine at the ends of the oligonucleotides and in the oligonucleotides proper to thymidine dimer (Nakane et al., Acta Histochem. Cytochem, 1987,20: 229-243). Polytene chromosomes will be prepared from the Drosophila strain Oregon R and will be spread over a light microscope glass slide.

The polytene chromosomes will be denatured, dehydrated, pretreated with proteinase A and hybridized with the thymidine dimerized oligonucleotides overnight at 37° C in 1.4 x SSC, 7% dextran sulfate, 35% N, N- dimethyl-formamide, and 0.6 mg/mL sonicated salmon DNA.

After washing the chromosomes, the hybridized thymidine dimerized oligonucleotides will be immuno-reacted with horseradish peroxidase conjugated mouse monoclonal anti- thymidine dimer. The sites of horseradish peroxidase will be visualized by an enzyme histochemistry technique using 3,3'-diaminobenzidine and hydrogen peroxide as chromogen and substrate, respectively. Finally the chromosomes will be stained with Giesma stain and embedded in Permount. The slides will be examined under a light microscope. The stained chromosomes will be evaluated for signal intensity and distribution over the chromosomes.

As used herein, the term"about"means within 10% of ; i. e.,"about"a 30 nucleotide oligonucleotide means from 27 to 33 nucleotides, etc.

In a further aspect of this invention, each different 30-mer oligonucleotide will be extended to 50-mer by an addition of 20 nucleotides whose sequences are complementary to various species specific nucleotide sequences found in common bacteria. The 50-mer oligonucleotides will be hybridized to the Drosophila chromosomes as above. DNA isolated from the cultured bacteria will be irradiated by ultraviolet light to form thymidine dimers within the bacterial DNA. The thymidine dimerized bacterial DNA will be heat denatured

and then hybridized with the 20-mer segment of the 50-mer oligonucleotides that have been hybridized to the chromosome through the 30-mer segment thereof. After washing the chromosomes, the hybridized thymidine dimerized probes will be immuno-reacted with horseradish peroxidase conjugated mouse monoclonal anti-thymidine dimer. The sites of horseradish peroxidase will be visualized using enzyme histochemistry techniques with 3,3'-diaminobenzidine and hydrogen peroxide as chromogen and substrate, respectively.

Finally the chromosomes will be stained with Giesma stain and embedded in Permount.

Stained areas on the chromosomes will correspond to sites where the 20-mer segment of the 50-mer has hybridized to the DNA sequence of the chromosome and the extended 20-mer has hybridized to the bacterial DNA, the 20-mer therefore being the complement of the nucleotide sequence of the bacterial DNA. The stained chromosomes will be evaluated for signal intensity and distribution over the chromosomes.

In a further aspect of this invention, each different 30-mer oligonucleotides with nucleotide sequences complementary to a portion of the chromosomes will be linked to mouse monoclonal antibodies. The 30-mer oligonucleotides linked to antibodies will then be hybridized to the heat denatured Drosophile chromosomes. The conditions of the hybridization may require some modifications such as change in the melting temperature (Tm) so that during the hybridization the immunoreactivity of the antibodies will not be drastically altered. A solution that contains antigens to the linked antibodies will be reacted and washed. The antigen that reacted with the antibody will be reacted with peroxidase- labeled polyclonal rabbit antibody against the antigen. After washing the chromosomes, the signal will be developed as described above. Stained regions on the chromosome will correspond to sites where the DNA sequence of the chromosome is complementary to the 30- mer portion of the oligonucleotide probe and where the probe has linked to antibodies that react with the added antigen. The stained chromosomes will be evaluated for signal intensity and distribution over the chromosomes.

CONCLUSION Thus, it will be appreciated that the methods and devices herein will provide for the effective, economical, easily carried out detection of substances in mixtures which may be

simple or complex through the use of known nucleotide sequences of naturally occurring chromosomes to form arrays of molecular probes capable of specifically binding the substances at pre-determined sites on the chromosome from which the identity of the substances can be determined.

Although certain embodiments and examples have been used to describe the present invention, it will be apparent to those skilled in the art that changes to the embodiments and examples shown may be made without departing from the scope and spirit of the invention.

Other embodiments are within the following claims.