GB2396352A

GB2396352A - Human genome-derived single exon nucleic acid probes

Info

Publication number: GB2396352A
Application number: GB0406170A
Authority: GB
Inventors: Sharron Gaynor Penn; David Kagen Hanzel; Wensheng Chen; David Russell Rank
Original assignee: Aeomica Inc
Current assignee: Aeomica Inc
Priority date: 2000-02-04
Filing date: 2001-01-30
Publication date: 2004-06-23
Anticipated expiration: 2021-01-30
Also published as: GB2396352B; GB0406170D0

Abstract

A single exon nucleic acid microarray comprises a plurality of specified single exon nucleic acid probes for measuring gene expression in a sample derived from human bone marrow. Also described are single exon nucleic acid probes expressed in the bone marrow and their use in methods for detecting gene expression.

Description

GB 2396352 A continuation (72) Inventor(s): Sharron Gaynor Penn David

Kagen Hanzel Wensheng Chen David Russell Rank (74) Agent and/or Address for Service: Amersham PLC Amersham Place, LITTLE CHALFONT, Buckinghamshire, HP7 BOA, United Kingdom

PB0004-W06-GB

HUMAN GENOME-DERIVED SINGLE EXON NUCLEIC ACID PROBES USEFUL

FOR ANALYSIS OF GENE EXPRESSION IN HUMAN BONE MARROW

REFERENCE TO SEQUENCE LISTING AND INCORPORATION BY

REFERENCE THEREOF

The present application includes a Sequence Listing in 20 electronic format, on a single CD-R disc containing a file named pto_BONE_MARROW.txt, created 24 January 2001, having 26,421,347 bytes. The Sequence Listing contained in said file on said disc is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to genome-derived single exon microarrays useful for verifying the expression 30 of regions of genomic DNA predicted to encode protein. In particular, the present invention relates to unique genome-

derived single exon nucleic acid probes expressed in human bone marrow and single exon nucleic acid microarrays that include such probes.

PB0004-W06-GB

Background of the Invention

For almost two decades following the invention of general techniques for nucleic acid sequencing, Sanger et al., Proc. Natl. Acad. Sci. USA 70(4) :1209-13 (1973); 5 Gilbert et al., Proc. Natl. Acad. Sci. USA 70(12):35814 (1973), these techniques were used principally as tools to further the understanding of proteins - known or suspected - about which a basic foundation of biological knowledge had already been built. In many cases, the 10 cloning effort that preceded sequence identification had been both informed and directed by that antecedent biological understanding.

For example, the cloning of the T cell receptor for antigen was predicated upon its known or suspected cell 15 type-specific expression, by its suspected membrane association, and by the predicted assembly of its gene via T cell-specific somatic recombination. Subsequent sequencing efforts at once confirmed and extended understanding of this family of proteins. Hedrick et al., 20 Nature 308(5955):153-8 (1984).

More recently, however, the development of high throughput sequencing methods and devices, in concert with large public and private undertakings to sequence the human and other genomes, has altered this investigational 25 paradigm: today, sequence information often precedes understanding of the basic biology of the encoded protein product. One of the approaches to large-scale sequencing is predicated upon the proposition that expressed 30 sequences - that is, those accessible through isolation of mRNA - are of greatest initial interest. This "expressed sequence tag" ("EST") approach has already yielded vast amounts of sequence data (see for example Adams et al., Science 252:1651 (1991); Williamson, Drug Discov. Today 35 4:115 (1999)). For nucleic acids sequenced by this

PB0004-W06-GB

approach, often the only biological information that is known a priori with any certainty is the likelihood of biologic expression itself. By virtue of the species and tissue from which the mRNA had originally been obtained, 5 most such sequences are also annotated with the identity of the species and at least one tissue in which expression appears likely.

More recently, the pace of genomic sequencing has accelerated dramatically. When genomic DNA serves as the lo initial substrate for sequencing efforts, expression cannot be presumed; often the only a priori biological information about the sequence includes the species and chromosome (and perhaps chromosomal map location) of origin.

With the ever-accelerating pace of sequence 15 accumulation by directed, EST, and genomic sequencing approaches - and in particular, with the accumulation of sequence information from multiple genera, from multiple species within genera, and from multiple individuals within a species there is an increasing need for methods that 20 rapidly and effectively permit the functions of nucleic sequences to be elucidated. And as such functional information accumulates, there is a further need for methods of storing such functional information in meaningful and useful relationship to the sequence itself; 2s that is, there is an increasing need for means and apparatus for annotating raw sequence data with known or predicted functional information.

Although the increase in the pace of genomic sequencing is due in large part to technological changes in 30 sequencing strategies and instrumentation, Service, Science 280:995 (1998); Pennisi, Science 283: 1822-1823 (1999), there is an important functional motivation as well.

While it was understood that the EST approach would rarely be able to yield sequence information about 35 the noncoding portions of the genome, it now also appears

PB0004-W06-GB

the EST approach is capable of capturing only a fraction of a genome's actual expression complexity.

For example, when the C. elegant genome was fully sequenced, gene prediction algorithms identified over 5 19,000 potential genes, of which only 7,000 had been found by EST sequencing. C. elegant Sequencing Consortium, Science 282:2012 (1998). Analogously, the recently completed sequence of chromosome 2 of Arabidopsis predicts over 4000 genes, Lin et al., Nature, 402:761 (1999), of 10 which only about 6% had previously been identified via EST sequencing efforts. Although the human genome has the greatest depth of EST coverage, it is still woefully short of surrendering all of its genes. One recent estimate suggests that the human genome contains more than 146,000 15 genes, which would at this point leave greater than half of the genes undiscovered. It is now predicted that many genes, perhaps 20 to 50%, will only be found by genomic sequencing. There is, therefore, a need for methods that 20 permit the functional regions of genomic sequence - and most importantly, but not exclusively, regions that function to encode genes - to be identified.

Much of the coding sequence of the human genome is not homologous to known genes, making detection of open 25 reading frames ("ORFs") and predictions of gene function difficult. Computational methods exist for predicting coding regions in eukaryotic genomes. Gene prediction programs such as GRAIL and GRAIL II, Uberbacher et al., Proc. Natl. Acad. Sci. USA 88(24):11261-5 (1991); Xu et 30 al., Genet. Eng. 16:241-53 (1994); Uberbacher et al., Methods Enzymol. 266:259-81 (1996); GENEFINDER, Solovyev et al., Nucl. Acids. Res. 22:5156-63 (1994); Solovyev et al., Ismb 5:294-302 (1997); and GENESCAN, Burge et al., J. Mol. Biol. 268:7894 (1997), predict many putative genes without 35 known homology or function. Such programs are known,

PB0004-W06-GB

however, to give high false positive rates. Burset et al., Genomics 34:353-367 (1996). Using a consensus obtained by a plurality of such programs is known to increase the reliability of calling exons from genomic sequence.

5 Ansari-Lari et al., Genome Res. 8(1):29-40 (1998) Identification of functional genes from genomic data remains, however, an imperfect art. For example, in reporting the full sequence of human chromosome 21, the Chromosome 21 Mapping and Sequencing Consortium reports 10 that prior bioinformatic estimates of human gene number may need to be revised substantially downwards. Nature 405:311-199 (2000); Reeves, Nature 405:283-284 (2000).

Thus, there is a need for methods and apparatus that permit the functions of the regions identified 15 bioinformatically - and specifically, that permit the expression of regions predicted to encode protein - readily to be confirmed experimentally.

Recently, the development of nucleic acid microarrays has made possible the automated and highly 20 parallel measurement of gene expression. Reviewed in Schena (ed.), DNA Microarrays: A Practical Approach (Practical Approach Series), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1 - 60 (1999); Schena (ed.), Microarray Biochip: Tools and 25 Technology, Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376).

It is common for microarrays to be derived from cDNA/EST libraries, either from those previously described in the literature, such as those from the I.M.A.G.E.

30 consortium, Lennon et al., Genomics 33(1):151-2 (1996), or from the construction of "problem specific" libraries targeted at a particular biological question, R.S. Thomas et al., Cancer Res. (in press). Such microarrays by definition can measure expression only of those genes found 35 in EST libraries, and thus have not been useful as probes

PB0004-W06-GB

for genes discovered solely by genomic sequencing.

The utility of using whole genome nucleic acid microarrays to answer certain biological questions has been demonstrated for the yeast Saccharomyces cerevisiae. De 5 Risi et al., Science 278:680 (1997). The vast majority of yeast nuclear genes, approximately 95% however, are single exon genes, i. e., lack introns, Lopez et al., RNA 5:1135-

1137 (1999); Goffeau et al., Science 274:563-67 (1996), permitting coding regions more readily to be identified.

10 Whole genome nucleic acid microarrays have not generally been used to probe gene expression from more complex eukaryotic genomes, and in particular from those averaging more than one intron per gene.

Because bone marrow is the tissue in which blood 15 cells originate, diseases of the bone marrow are a significant cause of human morbidity and mortality.

Increasingly, genetic factors are being found that contribute to predisposition, onset, and/or aggressiveness of most, if not all, of these diseases. Although mutations 20 in single genes have in some cases been identified as causal - notably in the thalassemias and sickle cell anemia - disorders of the bone marrow are, for the most part, believed to have polygenic etiologies. There is a need for methods and apparatus that permit prediction, diagnosis and 25 prognosis of diseases of the bone marrow, particularly those diseases with polygenic etiology.

Summary of the Invention

30 The present invention solves these and other problems in the art by providing methods and apparatus for predicting, confirming, and displaying functional information derived from genomic sequence. The present invention also provides apparatus for verifying the 35 expression of putative genes identified within genomic

PB0004-W06-GB

sequence. In particular, the invention provides novel genome-derived single exon nucleic acid microarrays useful for verifying the expression of putative genes identified 5 within genomic sequence.

The present invention also provides compositions and kits for the ready production of nucleic acids identical in sequence to, or substantially identical in sequence to, probes on the genome-derived single exon 10 microarrays of the present invention.

Accordingly, in a first aspect of the invention, there is provided a spatially-addressable set of single exon nucleic acid probes for measuring gene expression in a sample derived from human bone marrow, comprising a 15 plurality of single exon nucleic acid probes according to any one of the nucleotide sequences set out in SEQ ID NOs: 1 - 13,114 or a complementary sequence, or a portion of such a sequence.

By plurality is meant at least two, suitably at 20 least 20, most suitably at least 100, preferably at least 1000 and, most preferably, upto 5000.

In one embodiment of the first aspect, each of said plurality of probes is separately and addressably amplifiable. 25 In an alternative embodiment, each of said plurality of probes is separately and addressably isolatable from said plurality.

In a preferred embodiment, each of said plurality of probes is amplifiable using at least one common primer.

30 Preferably, each of said plurality of probes is amplifiable using a first and a second common primer.

In yet another embodiment, said set of single exon nucleic acid probes comprises between 50 - 20,000 probes, for example, 50 - 5000.

35 Suitably, said set of single exon nucleic acid

PB0004-WO6-GB

probes comprises at least 50 - 1000 discrete single exon nucleic acid probes having a sequence as set out in any of SEQ ID NOS.: 1 - 26,012 or a complimentary sequence, or a portion of such a sequence.

5 Preferably, the average length of the single exon nucleic acid probes is between 200 and 500 bp. It is preferred that the average length should be at least 200bp, suitably at least 250bp, most suitably at least 300bp, preferably at least 400bp and, most preferably, 500 bp.

10 In another embodiment, the single exon nucleic acid probes lack prokaryotic and bacteriophage vector sequence. It is preferred that at least 50%, suitably at least 60%, most suitably at least 70%, preferably at least 75%, more preferably at least 80, 85, 90, 95 or 99% of said 15 single exon nucleic acid probes lack prokaryotic and bacteriophage vector sequence.

In another preferred embodiment, said single exon nucleic acid lack homopolymeric stretches of A or T. It is preferred that at least 50%, suitably at least 60%, most 20 suitably at least 70%, preferably at least 75%, more preferably at least 80, 85, 90, 95 or 99% of said single exon nucleic acid probes lack homopolymeric stretches of A or T. Preferably, a spatially-addressable set of single 25 exon nucleic acid probes in accordance with the first aspect of the invention is is addressably disposed upon a substrate. Suitable substrates include a filter membrane which may, preferably, be nitrocellulose or nylon. The 30 nylon may preferably, be positively-charged. Other suitable substrates include glass, amorphous silicon, crystalline silicon, and plastic. Further suitable materials include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, 35 polytetrafluoroethylene, polystyrene, polycarbonate,

PB0004-W06-GB

polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, and mixtures thereof.

In a second aspect of the invention, there is provided a microarray comprising a spatially addressable 5 set of single exon nucleic acid probes in accordance with the first aspect of the invention.

In one embodiment, a genome-derived single-axon microarray is packaged together with such an ordered set of amplifiable probes corresponding to the probes, or one or 10 more subsets of probes, thereon. In alternative embodiments, the ordered set of amplifiable probes is packaged separately from the genome-derived single exon microarray. In another aspect, the invention provides genome 15 derived single exon nucleic acid probes useful for gene expression analysis, and particularly for gene expression analysis by microarray. In particular embodiments of this aspect, the present invention provides human single-axon probes that include specifically-hybridizable fragments of 20 SEQ ID Nos. 13,115 - 26,012, wherein the fragment hybridizes at high stringency to an expressed human gene.

In particular embodiments, the invention provides single exon probes comprising SEQ ID Nos. 1 - 13,114.

Accordingly, in a third aspect of the invention, 25 there is provided a single exon nucleic acid probe for measuring human gene expression in a sample derived from human bone marrow which is a nucleic acid molecule comprising a nucleotide sequence as set out in any of SEQ ID NOs.: 1 - 13, 114 or a complementary sequence or a 30 fragment thereof wherein said probe hybridizes at high stringency to a nucleic acid expressed in the human bone marrow. In one embodiment, a single exon nucleic acid probe in accordance with the third aspect comprises a 35 nucleotide sequence as set out in any of SEQ ID NOs.:

PB0004-W06-GB

13,115 - 26,012 or a complementary sequence or a fragment thereof. In a fourth aspect of the invention, there is provided a single exon nucleic acid probe for measuring 5 human gene expression in a sample derived from human bone marrow which is a nucleic acid molecule having a sequence encoding a peptide comprising a peptide sequence as set out in any of SEQ ID NOS.: 26,013 - 38,628 or a complementary sequence or a fragment thereof wherein said probe lo hybridizes at high stringency to a nucleic acid expressed in the human bone marrow.

Preferably, a single exon nucleic acid probe in accordance with the third or fourth aspects of the invention comprises between at least 15 and 50 contiguous 15 nucleotides of said SEQ ID NO:. It is preferred that the single exon nucleic acid probe comprises at least 15, suitably at least 20, more suitably at least 25 or preferably at least 50 contiguous nucleotides of said SEQ ID NO:.

20 In another preferred embodiment, a single exon nucleic acid probe in accordance with the third or fourth aspects of the invention is between 3kb and 25kb in length.

It is preferred that said probe is no more than 3kb, suitably no more than 5kb, more suitably no more than lOkb, 25 preferably 15kb, more preferably 20kb or, most preferably, no more than 2Okb in length.

Preferably, a single exon nucleic acid probe in accordance with either the fifth or sixth aspect of the invention is DNA, preferably single- stranded DNA, RNA or 30 PNA.

In another embodiment of either the third or fourth aspect of the invention, a single exon nucleic acid probe is detectably labeled. Suitable detectable labels include a radionuclide, a fluorescent label or a first 35 member of a specific binding pair. Suitable fluorescent

PB0004-W06-GB

- labels include dyes such as cyanine dyes, preferably Cy3 and Cy5 although other suitable dyes will be known to those skilled in the art.

In a particularly preferred embodiment, a single 5 exon nucleic acid probe in accordance with either the third or fourth aspect of the invention lacks prokaryotic and bacteriophage vector sequence. In yet another embodiment, a single exon nucleic acid probe in accordance with either the third or fourth aspect of the invention lacks 10 homopolymeric stretches of A or T. In a fifth aspect of the invention, there is provided an amplifiable nucleic acid composition, . comprising: the single exon nucleic acid probe in accordance 15 with either of the third or fourth aspects of the invention; and at least one nucleic acid primer; wherein said at least one primer is sufficient to prime enzymatic amplification of said probe.

In an sixth aspect of the invention, there is 20 provided a method of measuring gene expression in a sample derived from human bone marrow, comprising: contacting the single exon microarray in accordance with the second aspect of the invention, with a first collection of detectably labeled nucleic acids, said 25 first collection of nucleic acids derived from mRNA of human bone marrow; and then measuring the label detectably bound to each probe of said microarray.

In a seventh aspect of the invention, there is 30 provided a method of identifying exons in a eukaryotic genome, comprising: algorithmically predicting at least one exon from genomic sequence of said eukaryote; and then detecting specific hybridization of detectably 35 labeled nucleic acids to a single exon probe,

PB0004-W06-GB

Hi. wherein said detectably labeled nucleic acids are derived from mRNA from the bone marrow of said eukaryote, said probe is a single exon probe having a fragment identical in sequence to, or complementary in sequence to, 5 said predicted exon, said probe is included within a single exon microarray in accordance with the first aspect of the invention, and said fragment is selectively hybridizable at high stringency.

In a eighth aspect of the invention, there is lo provided a method of assigning exons to a single gene, . comprising: identifying a plurality of exons from genomic sequence in accordance with the seventh aspect of the invention; and then 15 measuring the expression of each of said exons in a plurality of tissues and/or cell types using hybridization to single exon microarrays having a probe with said exon, wherein a common pattern of expression of said 20 exons in said plurality of tissues and/or cell types indicates that the exons should be assigned to a single gene. In an ninth aspect of the invention, there is provided a nucleic acid sequence as set out in any of SEQ 25 ID NOs: 1 - 26012 wherein said sequence encodes a peptide.

In a tenth aspect of the invention, there is provided a peptide encoded by a sequence comprising a sequence as set out in any of SEQ ID NOs: 13, 115 - 26,012, or a complementary sequence or coding portion thereof.

30 In a preferred embodiment, a peptide may be encoded by a sequence comprising a sequence set out in any of SEQ ID NOS.: 1 - 13,114.

In a further aspect, the invention provides peptides comprising an amino acid sequence translated from 35 the DNA fragments, said amino acid sequences comprising SEQ

PB0004-W06-GB

ID NOS.: 26,013 - 38,628.

Accordingly in a eleventh aspect of the invention there is provided a peptide comprising a sequence as set out in any of SEQ ID NOs: 26,013 38,628, or fragment 5 thereof.

In another aspect, the invention provides means for displaying annotated sequence, and in particular, for displaying sequence annotated according to the methods and apparatus of the present invention. Further, such display 10 can be used as a preferred graphical user interface for electronic search, query, and analysis of such annotated sequence. 15 Detailed Description of the Invention

Definitions As used herein, the term "microarray" and phrase "nucleic acid microarray" refer to a substrate-bound 20 collection of plural nucleic acids, hybridization to each of the plurality of bound nucleic acids being separately detectable. The substrate can be solid or porous, planar or non-planar, unitary or distributed.

As so defined, the term "microarray" and phrase 25 "nucleic acid microarray" include all the devices so called in Schena (ed.), DNA Microarrays: A Practical Approach (Practical Approach Series), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1 60 (1999); and Schena (ed.), Microarray Biochip: Tools and 30 Technology, Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376). As so defined, the term "microarray" and phrase "nucleic acid microarray'' further include substrate-bound collections of plural nucleic acids in which the nucleic acids are distributable 35 disposed on a plurality of beads, rather than on a unitary

PB0004-WO6-GB

planar substrate, as is described, inter alla, in Brenner et al., Proc. Natl. Acad. Sci. USA 97(4):166501670 (2000); in such case, the term "microarray" and phrase "nucleic acid microarray" refer to the plurality of beads in 5 aggregate.

As used herein with respect to a nucleic acid microarray, the term "probe" refers to the nucleic acid that is, or is intended to be, bound to the substrate; in such context, the term "target" thus refers to nucleic acid 10 intended to be bound thereto by Watson-Crick complementarily. As used herein with respect to solution phase hybridization, the term "probe" refers to the nucleic acid of known sequence that is detectably labeled.

As used herein, the expression "probe comprising 15 SEQ ID NO.", and variants thereof, intends a nucleic acid probe, at least a portion of which probe has either (i) the sequence directly as given in the referenced SEQ ID NO., or (ii) a sequence complementary to the sequence as given in the referenced SEQ ID NO., the choice as between sequence 20 directly as given and complement thereof dictated by the requirement that the probe hybridize to mRNA.

As used herein, the term "open reading frame" and the equivalent acronym "ORF" refer to that portion of an exon that can be translated in its entirety into a sequence 25 of contiguous amino acids i.e. a nucleic acid sequence that, in at least one reading frame, does not possess stop codons; the term does not require that the ORF encode the entirety of a natural protein.

As used herein, the term "amplicon" refers to a 30 PCR product amplified from human genomic DNA, containing the predicted exon.

As used herein the term "exon" refers to the consensus prediction of the various exon and gene predicting algorithms i.e. a nucleic acid sequence 35 bioinformatically predicted to encode a portion of a

PB0004-W06-GB

: natural protein.

As used herein, the term "peptide" refers to a sequence of amino acids. The sequences referred to as PEPTIDE SEQ ID NOS.: are the predicted peptide sequences 5 that would be translated from one of the exons, or a portion thereof set out in exon SEQ ID NOS.:. The codons encoding the peptide are wholly contained within the exon.

As used herein, a "portions" of a defined nucleotide sequence or sequences can be and, preferably, 10 are fragments unique to that sequence or to one or a combination of those sequences. A fragment unique to a nucleic acid molecule is one that is a signature for the larger nucleic acid molecule.

As used herein, the phrase "expression of a 15 probe" and its linguistic variants means that the ORE present within the probe, or its complement, is present within a target mRNA.

As used herein, "stringent conditions refers to parameters well known to those skilled in the art. When a 20 nucleic acid molecule is said to be hybridizable to another of a given sequence under "stringent conditions" it is meant that it is homologous to the given sequence.

As used herein, the phrase "specific binding pair" intends a pair of molecules that bind to one another 25 with high specificity. Binding pairs are said to exhibit specific binding when they exhibit avidity of at least 107, preferably at least 108, more preferably at least 109 liters/mole. Nonlimiting examples of specific binding pairs are: antibody and antigen; biotin and avidin; and 30 biotin and streptavidin.

As used herein with respect to the visual display of annotated genomic sequence, the term "rectangle" means any geometric shape that has at least a first and a second border, wherein the first and second borders each are 3s capable of mapping uniquely to a point of another visual

PB0004-W06-GB

A_ object of the display.

As used herein, a "Mondrian" means a visual display in which a single genomic sequence is annotated with predicted and experimentally confirmed functional 5 information.

Brief Description of the Drawings

10 The present invention is further illustrated with reference to the following non-limiting figures and examples in which: FIG. 1 illustrates a process for predicting functional regions from genomic sequence, confirming the 15 functional activity of such regions experimentally, and associating and displaying the data so obtained in meaningful and useful relationship to the original sequence data; FIG. 2 further elaborates that portion of the 20 process schematized in FIG. 1 for predicting functional regions from genomic sequence; FIG. 3 illustrates a Mondrian visual display; FIG. 4 presents a Mondrian showing a hypothetical annotated genomic sequence; 25 FIG. 5 is a histogram showing the distribution of ORF length and PCR products as obtained, with ORE length shown in black and PCR product length shown in dotted lines; FIG. 6 is a histogram showing the distribution, 30 among exons predicted according to the methods described, of expression as measured using simultaneous two color hybridization to a genome-derived single exon microarray.

The graph shows the number of sequence-verified products that were either not expressed ("0"), expressed in one or 35 more but not all tested tissues ("1" - "9"), or expressed

PB0004-W06-GB

in all tissues tested ("10"); FIG. 7 is a pictorial representation of the expression of verified sequences that showed expression with signal intensity greater than 3 in at least one 5 tissue, with: FIG. 7A showing the expression as measured by microarray hybridization in each of the 10 measured tissues, and the expression as measured "bioinformatically" byquery of EST, NR and SwissProt databases; with FIG. 7B showing the legend for display of physical expression 10 (ratio) in FIG. 7A; and with FIG. 7C showing the legend for scoring EST hits as depicted in FIG. 7A; FIG. 8 shows a comparison of normalized CY3 signal intensity for arrayed sequences that were identical to sequences in existing EST, NR and SwissProt databases or 15 that were dissimilar (unknown), where black denotes the signal intensity for all sequence-verified products with a BLAST Expect ("E") value of greater than le-30 (1 x 10-3 ) ("unknown") and a dotted line denotes sequence-verified spots with a BLAST expect ("E") value of less than le-30 (1 20 x 10-3 )("known"); FIG. 9 presents a Mondrian of BAC AC008172 (bases 25,000 to 130,000), containing the carbamyl phosphate synthetase gene (AF154830.1); and FIG. 10 is a Mondrian of BAC A049839.

Methods and Apparatus for Predicting, Confirming, Annotating, and Displaying Functional Regions From Genomic Sequence Data FIG. 1 is a flow chart illustrating in broad outline a process for predicting functional regions from genomic sequence, confirming and characterizing the functional activity of such regions experimentally, and 35 then associating and displaying the information so obtained

PB0004-W06-GB

-_\ in meaningful and useful relationship to the original sequence data.

The initial input into process lo of the present invention is drawn from one or more databases lOO 5 containing genomic sequence data. Because genomic sequence is usually obtained from subgenomic fragments, the sequence data typically will be stored in a series of records corresponding to these subgenomic sequenced fragments.

Some fragments will have been catenated to form larger 10 contiguous sequences ("contigs"); others will not. A finite percentage of sequence data in the database will typically be erroneous, consisting inter alla of vector sequence, sequence created from aberrant cloning events, sequence of artificial polylinkers, and sequence that was 15 erroneously read.

Each sequence record in database lOO will minimally contain as annotation a unique sequence identifier (accession number), and will typically be annotated further to identify the date of accession, 20 species of origin, and depositor. Because database lOO can contain nongenomic sequence, each sequence will typically be annotated further to permit query for genomic sequence.

Chromosomal origin, optionally with map location, can also be present. Data can be, and over time increasingly will 25 be, further annotated with additional information, in part through use of the present invention, as described below.

Annotation can be present within the data records, in information external to database lOO and linked to the records thereto, or through a combination of the two.

30 Databases useful as genomic sequence database lOO in the present invention include GenBank, and particularly include several divisions thereof, including the htgs(draft), NT (nucleotide, command line), and NR (nonredundant) divisions. GenBank is produced by the 3s National Institutes of Health and is maintained by the

PB0004-W06-GB

National Center for Biotechnology Information (NCBI).

Databases of genomic sequence from species other than human, such as mouse, rat, Arabidopsis, C. elegans, C. brigsii, Drosophila, zebra fish, and other higher 5 eukaryotic organisms will also prove useful as genomic sequence database 100.

Genomic sequence obtained by query of genomic sequence database 100 is then input into one or more processes 200 for identification of regions therein that 10 are predicted to have a biological function as specified by the user. Such functions include, but are not limited to, encoding protein, regulating transcription, regulating message transport after transcription into mRNA, regulating message splicing after transcription into mRNA, of 15 regulating message degradation after transcription into mRNA, and the like. Other functions include directing somatic recombination events, contributing to chromosomal stability or movement, contributing to allelic exclusion or X chromosome inactivation, and the like.

20 The particular genomic sequence to be input into process 200 will depend upon the function for which relevant sequence is to be identified as well as upon the approach chosen for such identification. Process step 200 can be iterated to identify different functions within a 25 given genomic region. In such case, the input often will be different for the several iterations.

Sequences predicted to have the requisite function by process 200 are then input into process 300, where a subset of the input sequences suitable for 30 experimental confirmation is identified. Experimental confirmation can involve physical and/or bioinformatic assay. Where the subsequent experimental assay is bioinformatic, rather than physical, there are fewer constraints on the sequences that can be tested, and in 35 this latter case therefore process 300 can output the

PB0004-W06-GB

entirety of the input sequence.

The subset of sequences output from process 300 is then used in process 400 for experimental verification and characterization of the function predicted in 5 process 200, which experimental verification can, and often will, include both physical and bioinformatic assay.

Process 500 annotates the sequence data with the functional information obtained in the physical and/or bioinformatic assays of process 400. Such annotation can 10 be done using any technique that usefully relates the functional information to the sequence, as, for example, by incorporating the functional data into the sequence data record itself, by linking records in a hierarchical or relational database, by linking to external databases, by a 15 combination thereof, or by other means well known within the database arts. The data can even be submitted for incorporation into databases maintained by others, such as GenBank, which is maintained by NCBI.

As further noted in FIG. l, additional annotation 20 can be input into process 500 from external sources 600.

The annotated data is then displayed in process 800, either before, concomitantly with, or after optional storage 700 on nontransient media, such as magnetic disk, optical disc, magnetooptical disk, flash memory, or the 25 like.

FIG. l shows that the experimental data output from process 400 can be used in each preceding step of process lo: e.g., facilitating identification of functional sequences in process 200, facilitating identification of an 30 experimentally suitable subset thereof in process 300, and facilitating creation of physical and/or informational substrates for, and performance of subsequent assay, of functional sequences in process 400.

Information from each step can be passed directly 35 to the succeeding process, or stored in permanent or

PB0004-WO6-GB

interim form prior to passage to the succeeding process.

Often, data will be stored after each, or at least a plurality, of such process steps. Any or all process steps can be automated.

5 FIG. 2 further elaborates the prediction of functional sequence within genomic sequence according to process 200.

Genomic sequence database 100 is first queried 20 for genomic sequence.

10 The sequence required to be returned by query 20 will depend, in the first instance, upon the function to be identified. For example, genomic sequences that function to encode protein can be identified inter alla using gene 15 prediction approaches, comparative sequence analysis approaches, or combinations of the two. In gene prediction analysis, sequence from one genome is input into process 200 where at least one, preferably a plurality, of algorithmic methods are applied to identify putative coding 20 regions. In comparative sequence analysis, by contrast, corresponding, e.g., syntenic, sequence from a plurality of sources, typically a plurality of species, is input into process 200, where at least one, possibly a plurality, of algorithmic methods are applied to compare the sequences 25 and identify regions of least variability.

The exact content of query 20 will also depend upon the database queried. For example, if the database contains both genomic and nongenomic sequence, perhaps derived from multiple species, and the function to be 30 determined is protein coding regions in human genomic sequence; the query will accordingly require that the sequence returned be genomic and derived from humans.

Query 20 can also incorporate criteria that compel return of sequence that meets operative requirements 35 of the subsequent analytical method. Alternatively, or in

- PB0004-WO6-GB

addition, such operative criteria can be enforced in subsequent preprocess step 24.

For example, if the function sought to be identified is protein coding, query 20 can incorporate 5 criteria that return from genomic sequence database 100 only those sequences present within contigs sufficiently long as to have obviated substantial fragmentation of any given exon among a plurality of separate sequence fragments. 10 Such criteria can, for example, consist of a required minimal individual genomic sequence fragment length, such as 10 kb, more typically 20 kb, 30 kb, 40kb, and preferably 50 kb or more, as well as an optional further or alternative requirement that sequence from any 15 given clone, such as a bacterial artificial chromosome ("BAC"), be presented in no more than a finite maximal number of fragments, such as no more than 20 separate pieces, more typically no more than 15 fragments, even more typically no more than about 10 - 12 fragments.

20 Results using the present invention have shown that genomic sequence from bacterial artificial chromosomes (BACs) is sufficient for gene prediction analysis according to the present invention if the sequence is at least 50 kb in length, and if additionally the sequence from any given 25 BAC is presented in fewer than 15, and preferably fewer than 10, fragments. Accordingly, query 20 can incorporate a requirement that data accessioned from BAC sequencing be in fewer than 15, preferably fewer than 10, fragments.

An additional criterion that can be incorporated 30 into the query can be the date, or range of dates, of sequence accession. Although the process has been described above as if genomic sequence database 100 were static, it is of course understood that the genomic sequence databases need not be static, and indeed are 35 typically updated on a frequent, even hourly, basis. Thus,

PB0004-W06-GB

as further described in Examples l and 2, infra, it is possible to query the database for newly added sequence, either newly added after an absolute date, or newly added relative to a prior analysis performed using the methods 5 and apparatus of the present invention. In this way, the process herein described can incorporate a dynamic, temporal component.

One utility of such temporal limitation is to identify, from newly accessioned genomic sequence, the 10 presence of novel genes, particularly those not previously identified by EST sequencing (or other sequencing efforts that are similarly based upon gene expression). As further described in Example l, such an approach has shown that newly accessioned human genomic sequence, when analyzed for 15 sequences that function to encode protein, readily identifies genes that are novel over those in existing EST and other expression databases. This makes the methods of the present invention extremely powerful gene discovery tools. And as would be appreciated, such gene discovery 20 can be performed using genomic sequence from species other than human.

If query 20 incorporates multiple criteria, such as above-described, the multiple criteria can be performed as a series of separate queries or as a single query, 25 depending in part upon the query language, the complexity of the query, and other considerations well known in the database arts.

If query 20 returns no genomic sequence meeting the query criteria, the negative result can be reported by 30 process 22, and process 200 (and indeed, entire process lo) ended 23, as shown. Alternatively, or in addition to report and termination of the initial inquiry, a new query 20 can be generated that takes into account the initial negative result.

35 When query 20 returns sequence meeting the query

PB0004-W06-GB

criteria, the returned sequence is then passed to optional preprocessing 24, suitable and specific for the desired analytical approach and the particular analytical methods thereof to be used in process 25.

5 Preprocessing 24 can include processes suitable for many approaches and methods thereof, as well as processes specifically suited for the intended subsequent analysis. Preprocessing 24 suitable for most approaches and 10 methods will include elimination of sequence irrelevant to, or that would interfere with, the subsequent analysis.

Such sequence includes repetitive sequence, such as Alu repeats and LINE elements, vector sequence, artificial sequence, such as artificial polylinkers, and the like.

15 Such removal can readily be performed by identification and subsequent masking of the undesired sequence.

Identification can be effected by comparing the genomic sequence returned by query 20 with public or private databases containing known repetitive sequence, 20 vector sequence, artificial sequence, and other artifactual sequence. Such comparison can readily be done using programs well known in the art, such as CROSS_MATCH, or by proprietary sequence comparison programs the engineering of which is well within the skill in the art.

25 Alternatively, or in addition, undesirable, including artifactual, sequence can be identified algorithmically without comparison to external databases and thereafter removed. For example, synthetic polylinker sequence can be identified by an algorithm that identifies 30 a significantly higher than average density of known restriction sites. As another example, vector sequence can be identified by algorithms that identify nucleotide or codon usage at variance with that of the bulk of the genomic sequence.

35 Once identified, undesired sequence can be

PB0004-W06-GB

removed. Removal can usefully be done by masking the undesired sequence as, for example, by converting the specific nucleotide references to one that is unrecognized by the subsequent bioinformatic algorithms, such as "X".

5 Alternatively, but at present less preferred, the undesired sequence can be excised from the returned genomic sequence, leaving gaps.

Preprocessing 24 can further include selection from among duplicative sequences of that one sequence of 10 highest quality. Higher quality can be measured as a lower percentage of, fewest number of, or least densely clustered occurrence of ambiguous nucleotides, defined as those nucleotides that are identified in the genomic sequence using symbols indicating ambiguity. Higher quality can 15 also or alternatively be valued by presence in the longest contig. Preprocessing 24 can, and often will, also include formatting of the data as specifically appropriate for passage to the analytical algorithms of process 25.

20 Such formatting can and typically will include, inter alla, addition of a unique sequence identifier, either derived from the original accession number in genomic sequence database 100, or newly applied, and can further include additional annotation. Formatting can include conversion 25 from one to another sequence listing standard, such as conversion to or from FASTA or the like, depending upon the input expected by the subsequent process.

Preprocessing, which can be optional depending upon the function desired to be identified and the 30 informational requirements of the methods for effecting such identification, is followed by sequence processing 25, where sequences with the desired function are identified within the genomic sequence.

As mentioned above, such functions can include, 35 but are not limited to, encoding protein, regulating

PB0004-W06-GB

transcription, regulating message transport after transcription into mRNA, regulating message splicing after transcription, of regulating message degradation, and the like. Other functions include directing somatic 5 recombination events, contributing to chromosomal stability or movement, contributing to allelic exclusion or X chromosome inactivation, or the like.

The methods of the present invention are particularly useful for gene discovery, that is, for 10 identifying, from genomic sequence, regions that function to encode genes, and in a particularly useful embodiment, for identifying regions that function to encode genes not hitherto identified by expression-based or directed cloning and sequencing. In conjunction with verification using the 15 novel single exon microarrays of the present invention, as further described below, the methods herein described become powerful gene discovery tools.

Accordingly, in a preferred embodiment of the present invention, process 25 is used to identify putative 20 coding regions. Two preferred approaches in process 25 for identifying sequence that encodes putative genes are gene prediction and comparative sequence analysis.

Gene prediction can be performed using any of a number of algorithmic methods, embodied in one or more 25 software programs, that identify open reading frames (ORFs) using a variety of heuristics, such as GRAIL, DICTION, and GENEFINDER. Comparative sequence analysis similarly can be performed using any of a variety of known programs that identify regions with lower sequence variability.

30 As further described in Example l, below, gene finding software programs yield a range of results. For the newly accessioned human genomic sequence input in Example l, for example, GRAIL identified the greatest percentage of genomic sequence as putative coding region, 35 2% of the data analyzed; GENEFINDER was second, calling 1%;

PB0004-W06-GB

and DICTION yielded the least putative coding region, with 0.8% of genomic sequence called as coding region.

Increased reliability can be obtained when consensus is required among several such methods. Although 5 discussed herein particularly with respect to exon calling, consensus among methods will in general increase reliability of predicting other functions as well.

Thus, as indicated by query 26, sequence processing 25, optionally with preprocessing 24, can be 10 repeated with a different method, with consensus among such iterations determined and reported in process 27.

Process 27 compares the several outputs for a given input genomic sequence and identifies consensus among the separately reported results. The consensus itself, as 15 well as the sequence meeting that consensus, is then stored in process 29a, displayed in process 29b, and/or output to process 300 for subsequent identification of a subset thereof suitable for assay.

Multiple levels of consensus can be calculated 20 and reported by process 27. For example, as further described in Example l, infra, process 27 can report consensus as between all specific pairs of methods of gene prediction, as consensus among any one or more of the pairs of methods of gene prediction, or as among all of the gene 25 prediction algorithms used. Thus, in Example l, process 27 reported that GRAIL and GENEFINDER programs agreed on 0.7% of genomic sequence, that GRAIL and DICTION agreed on 0.5% of genomic sequence, and that the three programs together agreed on 0.25% of the data analyzed. Put another way, 30 0.25% of the genomic sequence was identified by all three of the programs as containing putative coding region.

Furthermore, consensus can be required among different approaches to identifying a chosen function.

For example, if the function desired to be 35 identified is coding of protein sequence, and a first used

PB0004-W06-GB

approach to exon calling is gene prediction, the process can be repeated on the same input sequence, or subset thereof, with another approach, such as comparative sequence analysis. In such a case, where comparative 5 sequence analysis follows gene prediction, the comparison can be performed not only on genomic nucleic acid sequence, but additionally or alternatively can be performed on the predicted amino acid sequence translated from the ORFs prior identified by the gene prediction approach.

10 Although shown as an iterative process, the multiple analyses required to achieve consensus can be done in series, in parallel, or some combination thereof.

Predicted functional sequence, optionally representing a consensus among a plurality of methods and 15 approaches for determination thereof, is passed to process 300 for identification of a subset thereof for functional assay. In the preferred embodiment of the methods of the present invention, wherein the function sought to be 20 identified is protein coding, process 300 is used to identify a subset thereof suitable for experimental verification by physical and/or bioinformatic approaches.

For example, putative ORFs identified in process 200 can be classified, or binned, bioinformatically into 25 putative genes. This binning can be based inter alla upon consideration of the average number of axons/gene in the species chosen for analysis, upon density of exons that have been called on the genomic sequence, and other empirical rules. Thereafter, one or more among the gene 30 specific ORFs can be chosen for subsequent use in gene expression assay.

Where such subsequent gene expression assay uses amplified nucleic acid, considerations such as desired amplicon length, primer synthesis requirements, putative 35 exon length, sequence GC content, existence of possible

PB0004-W06-GB

secondary structure, and the like can be used to identify and select those ORFs that appear most likely successfully to amplify. Where subsequent gene expression assay relies upon nucleic acid hybridization, whether or not using 5 amplified product, further considerations involving hybridization stringency can be applied to identify that subset of sequences that will most readily permit sequence-

specific discrimination at a chosen hybridization and wash stringency. One particular such consideration is avoidance 10 of putative exons that span repetitive sequence; such sequence can hybridize spuriously to nonspecific message, reducing specific signal in the hybridization.

For bioinformatic assay, there are fewer constraints on the sequences that can be tested 15 experimentally, and in this latter case therefore process 300 can output the entirety of the input sequence.

The subset of sequences identified by process 300 as suitable for use in assay is then used in process 400 to create the physical and/or informational substrate for 20 experimental verification of the predictions made in process 200, and thereafter to assay those substrates.

As mentioned, the methods of the present invention are particularly useful for identifying potential coding regions within genomic sequence. In a preferred 25 embodiment of process 400, therefore, the expression of the sequences predicted to encode protein is verified. The combination of the predictive and experimental methods provides a powerful gene discovery engine.

Thus, in another aspect, the present invention 30 provides methods and apparatus for verifying the expression of putative genes identified within genomic sequence. In particular, the invention provides a novel method of verifying gene expression in which expression of predicted ORFs is measured and confirmed using a novel type of 35 nucleic acid microarray, the genome-derived single exon

PB0004-W06-GB

nucleic acid microarrays of the present invention.

Putative ORFs as predicted by a consensus of gene calling, particularly gene prediction, algorithms in process 200, and as further identified as suitable by s process 300, are amplified from genomic DNA using the polymerase chain reaction (PCR). Although PCR is conveniently used, other amplification approaches can also be used.

Amplification schemes can be designed to capture 10 the entirety of each predicted ORF in an amplicon with minimal additional (that is, intronic or intergenic) sequence. Because ORFs predicted from human genomic sequence using the methods of the present invention differ in length, such an approach results in amplicons of varying 15 length.

However, most predicted ORFs are shorter than 500 bp in length, and although amplicons of at least about 100 or 200 base pairs can be immobilized as probes on nucleic acid microarrays, early experimental results using the 20 methods of the present invention have suggested that longer amplicons, at least about 400 or 500 base pairs, are more effective. Furthermore, certain advantages derive from application to the microarray of amplicons of defined size.

Therefore, amplification schemes can 25 alternatively, and preferably, be designed to amplify regions of defined size, preferably at least about 300, 400 or 500 bp, centered about each predicted ORF. Such an approach results in a population of amplicons of limited size diversity, but that typically contain intronic and/or 30 intergenic nucleic acid in addition to putative ORF.

Conversely, somewhat fewer than 10% of ORFs predicted from human genomic sequence according to the methods of the present invention exceed 500 bp in length.

Portions of such extended ORFs, preferably at least about 35 300,400 or 500 bp in length, can be amplified. However; it

PB0004-W06-GB

has been discovered that the percentage success at amplifying pieces of such ORFs is low, and that such putative exons are more effectively amplified when larger fragments, at least about 1000 or 1500 bp, and even as 5 large as 2000 bp are amplified.

The putative ORFs selected in process 300 are thus input into one or more primer design programs, such as PRIMERS (available online for use at http://www-genome.wi.mit.edu/cgi-bin/primer/), with a goal 10 of amplifying at least about 500 base pairs of genomic sequence centered within or about ORFs predicted to be no more than about 500 bp, or at least about 1000 - 1500 bp of genomic sequence for ORFs predicted to exceed 500 bp in length, and the primers synthesized by standard techniques.

15 Primers with the requisite sequences can be purchased commercially or synthesized by standard techniques.

Conveniently, a first predetermined sequence can be added commonly to the ORF-specific 5' primer and a second, typically different, predetermined sequence 20 commonly added to each 3' ORF-unique primer. This serves to immortalize the amplicon, that is, serves to permit further amplification of any amplicon using a single set of primers complementary respectively to the common 5' and common 3' sequence elements. The presence of these 25 ''universals' priming sequences further facilitates later sequence verification, providing a sequence common to all amplicons at which to prime sequencing reactions. The common 5' and 3' sequences further serve to add a cloning site should any of the ORFs warrant further study.

30 Such predetermined sequence is usefully at least about 10, 12 or 15 nt in length, and usually does not exceed about 25 nt in length. The "universal" priming sequences used in the examples presented infra were each 16 nt long.

35 The genomic DNA to be used as substrate for

PB0004-W06-GB

amplification will come from the eukaryotic species from which the genomic sequence data had originally been obtained, or a closely related species, and can conveniently be prepared by well known techniques from 5 somatic or germline tissue or cultured cells of the organism. See, e. g., Short Protocols in Molecular Biology : A Compendium of Methods from Current Protocols in Molecular Biology, Ausubel et al. (eds.), 4th edition (April 1999), John Wiley & Sons (ISBN: 047132938X) and 10 Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd edition (December 1989), ColdSpring Harbor Laboratory Press (ISBN: 0879693096). Many such prepared genomic DNAs are available commercially, with the human genomic DNAs additionally having certification of donor informed 15 consent.

Although the intronic and intergenic material flanking putative coding regions in the amplicons could potentially interfere with hybridizations during microarray experiments, we have found, surprisingly, that differential 20 expression ratios are not significantly affected. Rather, the predominant effect of exon size is to alter the absolute signal intensity, rather than its ratio. Equally surprising, the art had suggested that single exon probes would not provide sufficient signal intensity for high 25 stringency hybridization analyses; we find that such probes not only provide adequate signal, but have substantial advantages, as herein described.

After partial purification, as by size exclusion spin column, with or without confirmation as to amplicon 30 quality as by gel electrophoresis, each amplicon (single exon probe) is disposed in an array upon a support substrate. Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are 35 well known in the art (Reviewed by Schena et al., see

PB0004-WO6-GB

above). Typically, the support substrate will be glass, although other materials, such as amorphous or crystalline silicon or plastics. Such plastics include 5 polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof, can 10 also be used. Typically, the support will be rectangular, although other shapes, particularly circular disks and even spheres, present certain advantages. Particularly advantageous alternatives to glass slides as support substrates for array of nucleic acids are optical discs, as 15 described in WO 98/12559.

The amplified nucleic acids can be attached covalently to a surface of the support substrate or, more typically, applied to a derivatized surface in a chaotropic agent that facilitates denaturation and adherence by 20 presumed noncovalent interactions, or some combination thereof. Robotic spotting devices useful for arraying nucleic acids on support substrates can be constructed using public domain specifications (The MGuide, version

25 2.0, http://cmgm.stanford.edu/pErown/mguide/index.html), or can conveniently be purchased from commercial sources (MicroArray GenII Spotter and MicroArray GenIII Spotter, Molecular Dynamics, Inc., Sunnyvale, CA). Spotting can also be effected by printing methods, including those using 30 ink jet technology.

As is well known in the art, microarrays typically also contain immobilized control nucleic acids.

For controls useful in providing measurements of background

signal for the genome-derived single exon microarrays of 35 the present invention, a plurality of E. cold genes can

PB0004-W06-GB

readily be used. As further described in Example l, 16 or 32 E. cold genes suffice to provide a robust measure of background noise in such microarrays.

As is well known in the art, the amplified 5 product disposed in arrays on a support substrate to create a nucleic acid microarray can consist entirely of natural nucleotides linked by phosphodiester bonds, or alternatively can include either nonnative nucleotides, alternative internucleotide linkages, or both, so long as 10 complementary binding can be obtained in the hybridization.

If enzymatic amplification is used to produce the immobilized probes, the amplifying enzyme will impose certain further constraints upon the types of nucleic acid analogs that can be generated.

15 Although particularly described herein as using high density microarrays constructed on planar substrates, the methods of the present invention for confirming the expression of ORFs predicted from genomic sequence can use any of the known types of microarrays, as herein defined, 20 including lower density planar arrays, and microarrays on nonplanar, nonunitary, distributed substrates.

For example, gene expression can be confirmed using hybridization to lower density arrays, such as those constructed on membranes, such as nitrocellulose, nylon, 25 and positively-charged derivatized nylon membranes.

Further, gene expression can also be confirmed using nonplanar, beadbased microarrays such as are described in Brenner e t al., Proc. Natl. Acad. Sci. USA 9 7(4):166501670 (2000); U.S. Patent No. 6,057,107; and U. S. Patent No. 30 5,736,330. In theory, a packed collection of such beads provides in aggregate a higher density of nucleic acid probe than can be achieved with spotting or lithography techniques on a single planar substrate.

Planar microarrays on solid substrates, however, 35 provide certain useful advantages, including high

PB0004-W06-GB

: throughput and compatibility with existing readers. For example, each standard microscope slide can include at least 1000, typically at least 2000, preferably 5000 and upto 10,000 - 50,000 or more nucleic acid probes of 5 discrete sequence. The number of sequences deposited will depend on their required application.

Each putative gene can be represented in the array by a single predicted ORF. Alternatively, genes can be represented by more than one predicted ORF. For 10 purposes of measuring differential splicing, more than one predicted ORF will be provided for a putative gene. And as is well known in the art, each probe of defined sequence, representing a single predicted ORF, can be deposited in a plurality of locations on a single microarray to provide 15 redundancy of signal.

The genome-derived single exon microarrays described above differ in several fundamental and advantageous ways from microarrays presently used in the gene expression art, including (1) those created by 20 deposition of mRNA-derived nucleic acids, (2) those created by in situ synthesis of oligonucleotide probes, and (3) those constructed from yeast genomic DNA.

Most nucleic acid microarrays that are in use for study of eukaryotic gene expression have as immobilized 25 probes nucleic acids that are derived either directly or indirectly - from expressed message. As discussed above, it is common, for example, for such microarrays to be derived from cDNA/EST libraries, either from those previously described in the literature, see Lennon et al., 30 or from the de novo construction of "problem specific" libraries targeted at a particular biological question, R.S. Thomas et al., Cancer Res. (in press). Such microarrays are herein collectively denominated "EST microarrays". 35 Such EST microarrays by definition can measure

PB0004-W06-GB

: expression only of those genes found in EST libraries, shown herein to represent only a fraction of expressed genes. Furthermore, such libraries - and thus microarrays based thereupon - are biased by the tissue or cell type of 5 message origin, by the expression levels of the respective genes within the tissues, and by the ability of the message successfully to have been reverse-transcribed and cloned.

Thus, as further discussed in Example l, the methods of the present invention enable sequences that do 10 not appear in EST or other expression databases to be determined - subsequently arrayed for expression measurements could not, therefore, have been represented as probes on an EST microarray. And as further demonstrated in the examples, infra, the remaining population of genes 15 identified from genomic sequence by the methods of the present invention - that is, the one third of sequences that had previously been accessioned in EST or other expression databases - are biased toward genes with higher expression levels.

20 Representation of a message in an EST and/or cDNA library depends upon the successful reverse transcription, optionally but typically with subsequent successful cloning, of the message. This introduces substantial bias into the population of probes available for arraying in EST 25 microarrays.

In contrast, neither reverse transcription nor cloning is required to produce the probes arrayed on the genome-derived single exon microarrays of the present invention. And although the ultimate deposition of a probe 30 on the genome-derived single exon microarray of the present invention depends upon a successful amplification from genomic material, a priori knowledge of the sequence of the desired amplicon affords greater opportunity to recover any given probe sequence recalcitrant to amplification than is 35 afforded by the requirement for successful reverse

PB0004-W06-GB

transcription and cloning of unknown message in EST approaches. Thus, the genome-derived single exon microarrays of the present invention present a far greater diversity of 5 probes for measuring gene expression, with far less bias, than do EST microarrays presently used in the art.

As a further consequence of their ultimate origin from expressed message, the probes in EST microarrays often contain poly-A (or complementary polyT) stretches derived 10 from the poly-A tail of mature RNA. These homopolymeric stretches contribute to cross-hybridization, that is, to a spurious signal occasioned by hybridization to the homopolymeric tail of a labeled cDNA that lacks sequence homology to the gene-specific portion of the probe.

15 In contrast, the probes arrayed in the genome derived single exon microarrays of the present invention lack homopolymeric stretches derived from message polyadenylation, and thus can provide more specific signal.

* Typically, at least about 50, 60 or 75% of the probes on 20 the genomederived single exon microarrays of the present invention lack homopolymeric regions consisting of A or T. where a homopolymeric region is defined for purposes herein as stretches of 25 or more, typically 30 or more, identical nucleotides. 25 A further distinction, which also affects the specificity of hybridization, is occasioned by the typical derivation of EST microarray probes from cloned material.

Because much of the probe material disposed as probes on EST microarrays is excised or amplified from plasmid, 30 phage, or phagemid vectors, EST microarrays typically include a fair amount of vector sequence, more so when the probes are amplified, rather than excised, from the vector.

In contrast, the vast majority of probes in the genome-derived single exon microarrays of the present 35 invention contain no prokaryotic or bacteriophage vector

PB0004-W06-GB

sequence, having been amplified directly or indirectly from genomic DNA. Typically, therefore, at least about 50, 60, 70 or 80% or more of individual axon-including probes disposed on a genome-derived single exon microarray of the 5 present invention lack vector sequence, and particularly lack sequences drawn from plasmids and bacteriophage.

Preferably, at least about 85, 90 or more than 90% of exon-

including probes in the genome-derived single exon microarray of the present invention lack vector sequence.

10 With attention to removal of vector sequences through preprocessing 24, percentages of vector-free axon-including probes can be as high as 95 99%. The substantial absence of vector sequence from the genome-derived single exon microarrays of the present invention results in greater 15 specificity during hybridization, since spurious cross-

hybridization to a probe vector sequence is reduced.

As a further consequence of excision or amplification of probes from vectors in construction of EST microarrays, the probes arrayed thereon often contain 20 artificial sequence, derived from vector polylinker multiple cloning sites, at both 5' and 3' ends. The probes disposed upon the genome-derived single exon microarrays need have no such artificial sequence appended thereto.

As mentioned above, however, the ORF-specific 25 primers used to amplify putative ORFs can include artificial sequences, typically 5' to the ORFspecific primer sequence, useful for "universal" (that is, independent of ORF sequence) priming of subsequent amplification or sequencing reactions. When such 30 "universal" 5' and/or 3' priming sequences are appended to the amplification primers, the probes disposed upon the genome-derived single exon microarray will include artificial sequence similar to that found in EST microarrays. However, the genome-derived single exon 35 microarray of the present invention can be made without

PB0004-WO6-GB

_N such sequences, and if so constructed, presents an even smaller amount of nonspecific sequence that would contribute to nonspecific hybridization.

Yet another consequence of typical use of cloned 5 material as probes in EST microarrays is that such microarrays contain probes that result from cloning artifacts, such as chimeric molecules containing coding region of two separate genes. Derived from genomic material, typically not thereafter cloned, the probes of 10 the genome-derived single exon microarrays of the present invention lack such cloning artifacts, and thus provide greater specificity of signal in gene expression measurements. A further consequence of the cloned origin of 15 probes on many EST microarrays is that the individual probes often have disparate sizes, which can cause the optimal hybridization stringency to vary among probes on a single microarray. In contrast, as discussed above, the probes arrayed on the genome-derived single exon 20 microarrays of the present invention can readily be designed to have a narrow distribution in sizes, with the range of probe sizes no greater than about 10% of the average size, typically no greater than about 5% of the average probe size.

25 Because of their origin from fully- or partially spliced message, probes disposed upon EST arrays will often include multiple exons. The percentage of such exon spanning probes in an EST microarray can be calculated, on average, based upon the predicted number of axons/gene for 30 the given species and the average length of the immobilized probes. For human genes, the near-complete sequence of human chromosome 22, Dunham et al., Na Lure 402 (6761):489-95 (1999), predicts that human genes average 5.5 axons/gene.

Even with probes of 200 - 500 bp, the vast majority of 35 human EST microarray probes include more than one exon.

PB0004-W06-GB

: In contrast, by virtue of their origin from algorithmically identified ORFs in genomic sequence, the probes in the genome-derived single exon microarrays of the present invention can consist of individual exons. Thus, 5 in contrast to EST microarrays, at least about 50, 60, 70, 75, 80, 85, 95 or 99% of probes deposited in the genome-

derived microarray of the present invention consist of, or include, no more than one predicted ORF.

This provides the ability, not readily achieved 10 using EST microarrays, to use the genome-derived single exon microarrays of the present invention to measure tissue-specific expression of individual exons, which in turn allows differential splicing events to be detected and characterized, and in particular, allows the correlation of 15 differential splicing to tissue-specific expression patterns. Furthermore, the exons that are represented in EST microarrays are often biased toward the 3' or 5' end of their respective genes, since sequencing strategies used 20 for EST identification are so biased. In contrast, no such 3' or 5' bias necessarily inheres in the selection of exons for disposition on the genome-derived single exon microarrays of the present invention.

Conversely, the probes provided on the genome 25 derived single exon microarrays of the present invention typically, but need not necessarily, include intronic and/or intergenic sequence that is absent from EST microarrays, which are derived from mature mRNA.

Typically, at least about 50, 60, 70, 80 or 90% of the 30 axon-including probes on the genome-derived single exon microarrays of the present invention include sequence drawn from noncoding regions. As discussed above, the additional presence of noncoding region does not significantly interfere with measurement of gene expression, and provides 35 the additional opportunity to assay prespliced RNA, and

PB0004-W06-GB

: thus measure such phenomena such as nuclear export control.

The genome-derived single exon microarrays of the present invention are also quite different from in situ synthesis microarrays, where probe size is severely 5 constrained by inadequacies in the photolithographic synthesis process.

Typically, probes arrayed on in si tu synthesis microarrays are limited to a maximum of about 25 bp. As a well known consequence, hybridization to such chips must be 10 performed at low stringency. In order, therefore, to achieve unambiguous sequence-specific hybridization results, the in si to synthesis microarray requires substantial redundancy, with concomitant programmed arraying for each probe of probe analogues with altered 15 (i.e., mismatched) sequence.

In contrast, the longer probe length of the genome-derived single exon microarrays of the present invention allows much higher stringency hybridization and wash. Typically, therefore, axon-including probes on the 20 genome-derived single exon microarrays of the present invention average at least about loo, 200, 300, 400 or 500 bp in length. By obviating the need for substantial probe redundancy, this approach permits a higher density of probes for discrete exons or genes to be arrayed on the 2s microarrays of the present invention than can be achieved for in si tu synthesis microarrays.

A further distinction is that the probes in in situ synthesis microarrays typically are covalently linked to the substrate surface. In contrast, the probes disposed 30 on the genome-derived microarray of the present invention typically are, but need not necessarily be, bound noncovalently to the substrate.

Furthermore, the short probe size on in si tu microarrays causes large percentage differences in the 35 melting temperature of probes hybridized to their

PB0004-W06-GB

complementary target sequence, and thus causes large percentage differences in the theoretically optimum stringency across the array as a whole.

In contrast, the larger probe size in the 5 microarrays of the present invention create lower percentage differences in melting temperature across the range of arrayed probes.

A further significant advantage of the microarrays of the present invention over in situ 10 synthesized arrays is that the quality of each individual probe can be confirmed before deposition. In contrast, the quality of probes cannot be assessed on a probe-by-probe basis for the in si tu synthesized microarrays presently being used.

15 The genome-derived single exon microarrays of the present invention are also distinguished over, and present substantial benefits over, the genome-derived microarrays from lower eukaryotes such as yeast. Lashkari et al., Proc. Natl. Acad. Sci. USA 94:13057-13062 (1997).

20 Only about 220 - 250 of the 6100 or so nuclear genes in Saccharomyces cerevisiae - that is, only about 4 - 5% - have standard, spliceosomal, introns, Lopez et al., Nucl. Acids Res. 28:85-86 (2000); Spingola et al., RNA 5(2):221-34 (1999). Furthermore, the entire yeast genome 25 has already been sequenced. These two facts permit the ready amplification and disposition of single-ORF amplicons on such microarray without the requirement for antecedent use of gene prediction and/or comparative sequence analyses. 30 Thus, a significant aspect of the present invention is the ability to identify and to confirm expression of predicted coding regions in genomic sequence drawn from eukaryotic organisms that have a higher percentage of genes having introns than do yeast such as 3s Saccharomyces cerevisiae, particularly in genomic sequence

PB0004-W06-GB

drawn from eukaryotes in which at least about 10, 20 or 50% of proteinencoding genes have introns. In preferred embodiments, the methods and apparatus of the present invention are used to identify and confirm expression of 5 novel genes from genomic sequence of eukaryotes in which the average number of introns per gene is at least about one, two or three or more.

After the physical substrate is prepared, experimental verification of predicted function is 10 performed.

In a preferred embodiment of the present invention, where the function sought to be identified in genomic sequence is protein coding, experimental verification is performed by measuring expression of the 15 putative ORFs, typically through nucleic acid hybridization experiments, and in particularly preferred embodiments, through hybridization to genome-derived single exon microarrays prepared as above- described.

Expression is conveniently measured and expressed 20 for each probe in the microarray as a ratio of the expression measured concurrently in a plurality of mRNA sources, according to techniques well known in the microarray art, Reviewed in Schena et al., and as further described in Example 2, below. The mRNA source for the 2s reference against which specific expression is measured can be drawn from a homogeneous mRNA source, such as a single cultured cell-type, or alternatively can be heterogeneous, as from a pool of mRNA derived from multiple tissues and/or cell types, as further described in Example 2, infra.

30 mRNA can be prepared by standard techniques, see Ausubel et al. and Maniatis et al., or purchased commercially. The mRNA is then typically reverse transcribed in the presence of labeled nucleotides: the index source (that in which expression is desired to be 3s measured) is reverse transcribed in the presence of

PB0004-W06-GB

nucleotides labeled with a first label, typically a fluorophore (fluorochrome; fluor; fluorescent dye); the reference source is reverse transcribed in the presence of a second label, typically a fluorophore, typically 5 fluorometrically-distinguishable from the first label. As further described in Example 2, infra, Cy3 and Cy5 dyes prove particularly useful in these methods. After partial purification of the index and reference targets, hybridization to the probe array is conducted according to 10 standard techniques, typically under a coverelip.

After wash, microarrays are conveniently scanned using a commercial microarray scanning device, such as a Gen3 Scanner (Molecular Dynamics, Sunnyvale, CA). Data on expression is then passed, with or without interim storage, 15 to process 500, where the results for each probe are related to the original sequence.

Often, hybridization of target material to the genome-derived single exon microarray will identify certain of the probes thereon as of particular interest. Thus, it 20 is often desirable that the user be able readily to obtain sufficient quantities of an individual probe, either for subsequent arrayed deposition upon an additional support substrate, often as part of a microarray having a plurality of probes so identified, or alternatively or additionally 25 as a solitary solid-phase or solution- phase probe, for further use.

Thus, in another aspect, the present invention provides compositions and kits for the ready production of nucleic acids identical in sequence to, or substantially 30 identical in sequence to, probes on the genomederived single exon microarrays of the present invention.

In this aspect, a small quantity of each probe is disposed, typically without attachment to substrate, in a spatially-addressable ordered set, typically one per well 35 of a microtiter dish. Although a 96 well microtiter plate

PB0004-W06-GB

_N can be used, greater efficiency is obtained using higher density arrays, such as are provided by microtiter plates having 384, 864, 1536, 3456, 6144, or 9600 wells, and although microtiter plates having physical depressions 5 (wells) are conveniently used, any device that permits addressable withdrawal of reagent from fluidly-

noncommunicating areas can be used.

In this aspect of the invention, therefore, a fluidly noncommunicating addressable ordered set of 10 individual probes, corresponding to those on a genome-

derived single exon microarray, is provided, with each probe in sufficient quantity to permit amplification, such as by PCR. As earlier mentioned, the ORF-specific 5' primers used for genomic amplification can have a first 15 common sequence added thereto, and the ORF-specific 3' primers used for genomic amplification can have a second, different, common sequence added thereto, thus permitting, in this preferred embodiment, the use of a single set of 5' and 3' primers to amplify any one of the probes from the 20 amplifiable ordered set.

Each discrete amplifiable probe can also be packaged with amplification primers, solutes, buffers, etc., and can be provided in dry (e.g., lyophilized) form or wet, in the latter case typically with addition of 25 agents that retard evaporation.

In another aspect of the present invention, a genome-derived single-axon microarray is packaged together with such an ordered set of amplifiable probes corresponding to the probes, or one or more subsets of 30 probes, thereon. In alternative embodiments, the ordered set of amplifiable probes is packaged separately from the genome-derived single exon microarray.

In some embodiments, the microarray and/or ordered probe set are further packaged with recordable 35 media that provide probe identification and addressing

PB0004-W06-GB

J information, and that can additionally contain annotation information, such as gene expression data. Such recordable media can be packaged with the microarray, with the ordered probe set, or with both.

5 If the microarray is constructed on a substrate that incorporates recordable media, such as is described in international patent application no. WO 98/12559, then separate packaging of the genome- derived single exon microarray and the bioinformatic information is not 10 required.

The amount of amplifiable probe material should be sufficient to permit at least one amplification sufficient for subsequent hybridization assay.

Although the use of high density genome-derived 15 microarrays on solid planar substrates is presently a preferred approach for the physical confirmation and characterization of the expression of sequences predicted to encode protein, other types of microarrays (as herein defined) can also be used.

20 Furthermore, as earlier mentioned, experimental verification of the function predicted from genomic sequence in process 200 can be bioinformatic, rather than, or additional to, physical verification.

For example, where the function desired to be 25 identified is protein coding, the predicted ORFs can be compared bioinformatically to sequences known or suspected of being expressed.

Thus, the sequences output from process 300 (or process 200), can be used to query expression databases, 30 such as EST databases, SNP ("single nucleotide polymorphism") databases, known cDNA and mRNA sequences, SAGE (" serial analysis of gene expression") databases, and more generalized sequence databases that allow query for expressed sequences. Such query can be done by any 35 sequence query algorithm, such as BLAST ("basic local

PB0004-W06-GB

: alignment search tool"). The results of such query -

including information on identical sequences and information on nonidentical sequences that have diffuse or focal regions of sequence homology to the query sequence 5 can then be passed directly to process 500, or used to inform analyses subsequently undertaken in process 200, process 300, or process 400.

Experimental data, whether obtained by physical or bioinformatic assay in process 400, is passed to process 10 500 where it is usefully related to the sequence data itself, a process colloquially termed "annotation". Such annotation can be done using any technique that usefully relates the functional information to the sequence, as, for example, by incorporating the functional data into the - 15 record itself, by linking records in a hierarchical or relational

database, by linking to external databases, or by a combination thereof. Such database techniques are well within the skill in the art.

The annotated sequence data can be stored 20 locally, uploaded to genomic sequence database lOO, and/or displayed 800.

The methods and apparatus of the present invention rapidly produce functional information from genomic sequence. Coupled with the escalating pace at 25 which sequence now accumulates, the rapid-pace of sequence annotation produces a need for methods of displaying the information in meaningful ways.

FIG. 3 shows visual display 80 presenting a single genomic sequence annotated according to the present 30 invention. Because of its nominal resemblance to artistic works of Piet Mondrian, visual display 80 is alternatively described herein as a "Mondrian".

Each of the visual elements of display 80 is aligned with respect to the genomic sequence being 3s annotated (hereinafter, the "annotated sequence"). Given

PB0004-WO6-GB

the number of nucleotides typically represented in an annotated sequence, representation of individual nucleotides would rarely be readable in hard copy output of display 80. Typically, therefore, the annotated sequence 5 is schematized as rectangle 89, extending from the left border of display 80 to its right border. By convention herein, the left border of rectangle 89 represents the first nucleotide of the sequence and the right border of rectangle 89 represents the last nucleotide of the 10 sequence.

As further discussed below, however, the Mondrian visual display of annotated sequence can serve as a convenient graphical user interface for computerized representation, analysis, and query of information stored 15 electronically. For such use, the individual nucleotides can conveniently be linked to the X axis coordinate of rectangle 89. This permits the annotated sequence at any point within rectangle 89 readily to be viewed, either automatically - for example, by time-delayed appearance of 20 a small overlaid window upon movement of a cursor or other pointer over rectangle 89 - or through user intervention, as by clicking a mouse or other pointing device at a point in rectangle 89.

Visual display 80 is generated after user 25 specification of the genomic sequence to be displayed.

Such specification can consist of or include an accession

number for a single clone (e.g., a single BAC accessioned into GenBank), wherein the starting and stopping nucleotides are thus absolutely identified, or 30 alternatively can consist of or include an anchor or fulcrum point about which a chosen range of sequence is anchored, thus providing relative endpoints for the sequence to be displayed. For example, the user can anchor such a range about a given chromosomal map location, gene 35 name, or even a sequence returned by query for similarity

PB0004-W06-GB

) or identity to an input query sequence. When visual display 80 is used as a graphical user interface to computerized data, additional control over the first and last displayed nucleotide will typically be dynamically 5 selectable, as by use of standard zooming and/or selection tools. Field 81 of visual display 80 is used to present

the output from process 200, that is, to present the bioinformatic prediction of those sequences having the 10 desired function within the genomic sequence. Functional sequences are typically indicated by at least one rectangle 83 (83a, 83b, 83c), the left and right borders of which respectively indicate, by their X-axis coordinates, the starting and ending nucleotides of the region predicted to 15 have function.

Where a single bioinformatic method or approach identifies a plurality of regions having the desired function, a plurality of rectangles 83 is disposed horizontally in field 81. Where multiple methods and/or

20 approaches are used to identify function, each such method and/or approach can be represented by its own series of horizontally disposed rectangles 83, each such horizontally disposed series of rectangles offset vertically from those representing the results of the other methods and 25 approaches.

Thus, rectangles 83a in FIG. 3 represent the functional predictions of a first method of a first approach for predicting function, rectangles 83b represent the functional predictions of a second method and/or second 30 approach for predicting that function, and rectangles 83c represent the predictions of a third method and/or approach. Where the function desired to be identified is protein coding, field 81 is used to present the

35 bioinformatic prediction of sequences encoding protein.

PB0004-W06-GB

For example, rectangles 83a can represent the results from GRAIL or GRAIL II, rectangles 83b can represent the results from GENEFINDER, and rectangles 83c can represent the results from DICTION.

5 Optionally, and preferably, rectangles 83 collectively representing predictions of a single method and/or approach are identically colored and/or textured, and are distinguishable from the color and/or texture used for a different method and/or approach.

10 Alternatively, or in addition, the color, hue, density, or texture of rectangles 83 can be used further to report a measure of the bioinformatic reliability of the prediction. For example, many gene prediction programs will report a measure of the reliability of prediction.

15 Thus, increasing degrees of such reliability can be indicated, e.g., by increasing density of shading. Where display 80 is used as a graphical user interface, such measures of reliability, and indeed all other results output by the program, can additionally or alternatively be 20 made accessible through linkage from individual rectangles 83, as by timedelayed window ("tool tip" window), or by pointer (e.g., mouse)-activated link.

As earlier described, increased predictive reliability can be achieved by requiring consensus among 25 methods and/or approaches to determining function. Thus, field 81 can include a horizontal series of rectangles 83

that indicate one or more degrees of consensus in predictions of function.

Although FIG. 3 shows three series of 30 horizontally disposed rectangles in field 81, display 80

can include as few as one such series of rectangles and as many as can discriminately be displayed, depending upon the number of methods and/or approaches used to predict a given function. 3s Furthermore, field 81 can be used to show

PB0004-W06-GB

predictions of a plurality of different functions.

However, the increased visual complexity occasioned by such display makes more useful the ability of the user to select a single function for display. When display 80 is used as 5 a graphical user interface for computer query and analysis, such function can usefully be indicated and user-

selectable, as by a series of graphical buttons or tabs (not shown in FIG. 3).

Rectangle 89 is shown in FIG. 3 as including 10 interposed rectangle 84. Rectangle 84 represents the portion of annotated sequence for which predicted À functional information has been assayed physically, with the starting and ending nucleotides of the assayed material indicated by the X axis coordinates of the left and right 15 borders of rectangle 84. Rectangle 85, with optional inclusive circles 86 (86a, 86b, and 86c) displays the results of such physical assay.

Although a single rectangle 84 is shown in FIG. 3, physical assay is not limited to just one region of 20 annotated genomic sequence. It is expected that an increasing percentage of regions predicted to have function by process 200 will be assayed physically, and that display 80 will accordingly, for any given genomic sequence, have an increasing number of rectangles 84 and 85, representing 25 an increased density of sequence annotation.

Where the function desired to be identified is protein coding, rectangle 84 identifies the sequence of the probe used to measure expression. In embodiments of the present invention where expression is measured using 30 genome-derived single exon microarrays, rectangle 84 identifies the sequence included within the probe immobilized on the support surface of the microarray. As noted supra, such probe will often include a small amount of additional, synthetic, material incorporated during 35 amplification and designed to permit reamplification of the

PB0004-WO6-GB

probe, which sequence is typically not shown in display 80.

Rectangle 87 is used to present the results of bioinformatic assay of the genomic sequence. For example, where the function desired to be identified is protein 5 coding, process 400 can include bioinformatic query of expression databases with the sequences predicted in process 200 to encode exons. And as earlier discussed, because bioinformatic assay presents fewer constraints than does physical assay, often the entire output of process 200 10 can be used for such assay, without further subletting thereof by process 300. Therefore, rectangle 87 typically need not have separate indicators therein of regions submitted for bioinformatic assay; that is, rectangle 87 typically need not have regions therein analogous to 15 rectangles 84 within rectangle 89.

Rectangle 87 as shown in FIG. 3 includes smaller rectangles 880 and 88. Rectangles 880 indicate regions that returned a positive result in the bioinformatic assay, with rectangles 88 representing regions that did not return 20 such positive results. Where the function desired to be predicted and displayed is protein coding, rectangles 880 indicate regions of the predicted exons that identify sequence with significant similarity in expression databases, such as EST, SNP, SAGE databases, with 25 rectangles 88 indicating genes novel over those identified in existing expression data bases.

Rectangles 880 can further indicate, through color, shading, texture, or the like, additional information obtained from bioinformatic assay.

30 For example, where the function assayed and displayed is protein coding, the degree of shading of rectangles 880 can be used to represent the degree of sequence similarity found upon query of expression databases. The number of levels of discrimination can be 35 as few as two (identity, and similarity, where similarity

PB0004-W06-GB.

has a user-selectable lower threshold). Alternatively, as many different levels of discrimination can be indicated as can visually be discriminated.

Where display 80 is used as a graphical user 5 interface, rectangles 880 can additionally provide links directly to the sequences identified by the query of expression databases, and/or statistical summaries thereof.

As with each of the precedingly-discussed uses of display 80 as a graphical user interface, it should be understood 10 that the information accessed via display 80 need not be resident on the computer presenting such display, which often will be serving as a client, with the linked information resident on one or more remotely located servers. IS Rectangle 85 displays the results of physical assay of the sequence delimited by its left and right borders. Rectangle 85 can consist of a single rectangle, thus indicating a single assay, or alternatively, and 20 increasingly typically, will consist of a series of rectangles (85a, 85b, 85c) indicating separate physical assays of the same sequence.

Where the function assayed is gene expression, and where gene expression is assayed as herein described 25 using simultaneous two-color fluorescent detection of hybridization to genome-derived single exon microarrays, individual rectangles 85 can be colored to indicate the degree of expression relative to control. Conveniently, shades of green can be used to depict expression in the 30 sample over control values, and shades of red used to depict expression less than control, corresponding to the spectra of the Cy3 and Cy5 dyes conventionally used for respective labeling thereof. Additional functional information can be provided in the form of circles 86 (86a, 35 86b, 86c), where the diameter of the circle can be used to

PB0004-W06-GB

indicate expression intensity. As discussed infra, such relative expression (expression ratios) and absolute expression (signal intensity) can be expressed using normalized values.

5 Where display 80 is used as a graphical user interface, rectangle 85 can be used as a link to further information about the assay. For example, where the assay is one for gene expression, each rectangle 85 can be used to link to information about the source of the hybridized 10 mRNA, the identity of the control, raw or processed data from the microarray scan, or the like.

FIG. 4 is rendition of display 80 representing gene prediction and gene expression for a hypothetical BAC, showing conventions used in the Examples presented infra.

15 BAC sequence ("Chip seq.") 89 is presented, with the physically assayed region thereof (corresponding to rectangle 84 in FIG. 3) shown in white. Algorithmic gene predictions are shown in field 81, with predictions by

GRAIL shown, predictions by GENEFINDER, and predictions by 20 DICTION shown. Within rectangle 87, regions of sequence that, when used to query expression databases, return identical or similar sequences ("EST hit") are shown as white rectangles (corresponding to rectangles 880 in FIG. 3), gray indicates low homology, and black indicates 25 unknowns (where black and gray would correspond to rectangles 88 in FIG. 3).

Although FIGS. 3 and 4 show a single stretch of sequence, uninterrupted from left to right, longer sequences are usefully represented by vertical stacking of 30 such individual Mondrians, as shown in FIGS. 9 and 10.

Single Exon Probes Useful For Measuring Gene Expression The methods and apparatus of the present 35 invention rapidly produce functional information from

PB0004-W06-GB

) genomic sequence. Where the function to be identified is protein coding, the methods and apparatus of the present invention rapidly identify and confirm the expression of portions of genomic sequence that function to encode 5 protein. As a direct result, the methods and apparatus of the present invention rapidly yield large numbers of single-axon nucleic acid probes, the majority from previously unknown genes, each of which is useful for measuring and/or surveying expression of a specific gene in 10 one or more tissues or cell types.

It is, therefore, another aspect of the present invention to provide genome-derived single exon nucleic acid probes useful for gene expression analysis, and particularly for gene expression analysis by microarray.

15 Using the methods and genome-derived single-axon microarrays of the present invention, we have for example readily identified a large number of unique ORFs from human genomic sequence. Using single exon probes that encompass these ORFs, we have demonstrated, through-microarray 20 hybridization analysis, the expression of 13,114 of these ORFs in bone marrow.

As would immediately be appreciated by one of skill in the art, each single exon probe having demonstrable expression in bone marrow is currently 25 available for use in measuring the level of its ORF's expression in bone marrow.

Because bone marrow is the tissue in which blood cells originate, diseases of the bone marrow are a significant cause of human morbidity and mortality.

30 Increasingly, genetic factors are being found that contribute to predisposition, onset, and/or aggressiveness of most, if not all, of these diseases. Although mutations in single genes have in some cases been identified as causal - notably in the thalassemias and sickle cell anemia 35 - disorders of the bone marrow are, for the most part,

PB0004-W06-GB

believed to have polygenic etiologies.

For example, cancers that originate in the bone marrow and lymphatic tissues such as the lymphomas, leukemias, and myeloma have been recognized as a major 5 health concern. An estimated 632,000 Americans are presently living with lymphoma, leukemia or myeloma, and over 110,000 new cases are anticipated each year. The new cases alone account for 11% of all cancer cases reported in the United States.

10 Lymphoma is a general term for a group of cancers of lymphocytes that manifest in the tissues of the lymphatic system. Eventually, monoclonal proliferation crowds out healthy cells and creates tumors which enlarge lymph nodes. Approximately 450,000 members of the U.S. 15 population are living with lymphoma: 160,000 with Hodgkin disease (HD) and 290,000 with non-Hodgkin lymphoma.

Hodgkin disease (HD) is a specialized form of lymphoma, and represent about 8% of all lymphomas. HD can be distinguish in tissues by the presence of an abnormal 20 cell called the Reed-Sternberg cell. Incidence rates of HD are higher in adolescents and young adults, but HD is considered to be one of the most curable forms of cancer.

Symptoms of HD include painless welling of lymph glands, fatigue, recurrent high fever, sweating at night, skin 25 irritations and loss of weight.

Although an infectious etiology has been proposed to account for the disproportionate incidence of HD among siblings reared together particularly an association with Epstein Barr Virus (EBV) - multiple genetic contributions 30 have also been suggested.

As early as 1986, linkage to HLA was suggested, with Klitz et al., Am. J. Hum. Genet. 54: 497-505 (1994) reporting an overall association of the nodular sclerosing (NSHD) group with the HLA class II region. Results of the 35 study suggested that susceptibility to NSHD is influenced

PB0004-W06-GB

by more than 1 locus within the class II region. Through a literature search, Shugart and Collins (2000), Europ. J. Hum. Genet. 8: 460-463 (2000), performed a combined segregation and linkage analysis on 59 nuclear families 5 with HD and concluded that HD is most likely determined by both an HLA-associated major gene and other non-HLA genetic factors, in conjunction with environmental effects.

Non-Hodgkin lymphoma (NHL) is a malignant monoclinal proliferation of the lymphoid cells in the 10 immune system, including bone marrow, spleen, liver and GI tract. The pathologic classification of NHL continues to evolve, reflecting new insights into the cells of origin and the biologic bases of these heterogeneous diseases.

The course of NHL varies from indolent and initially well 15 tolerated to rapidly fatal. Furthermore, common clinical symptoms of NHL, but rare in HD, are congestion and edema of the face and neck and ureteral compression.

Non-Hodgkin lymphoma (NHL) has been linked to a variety of specific genetic defects, including 26 mutated 20 genes and at least 9 identified chromosomal translocations.

Among the mutated genes are: ALK (2p23); API2 (MIHC, cIAP2) (llq22-q23); API4 (survivin, S W)(17q25(?)); ATM (ATA, ATC) (llq22.3); BCL1 (llql3.3); BCL10 (CLAP, CIPER)(lp22); BCL2 (18q21.3); BCL6 (LAZ3,ZNF51) (3q27); BLYM (lp32); BMI1 25 (lOpl3); CCND1 (DllS287E, Cyclin D,PRADl)(llql3); CD44 (MDU3, HA, MDU2)(llpter-pl3); FRAT1 (lOq23-q24(?)); FRAT2 (GBP)(10(?)); IL6 (IFNB2)(7p21); 1RF4 (MUM1, LSIRF) (6p25 p23); LCP1 (PLS2)(13ql4.1-ql4. 3); MALT1 (MLT)(18q21); MUC1 (PUM,PEM)(lq21); MYBL1 (AMYB, A-MYB)(8q22); MYC (CMYC, C 30 MYC)(8q24.12-q24.13); NBSl(8q21); NPM1 (B23)(5q35); PCNA (2Opl2); TIAM1 (2lq22.1); and TP53 (p53, P53)(17ql3.1).

Among the chromosomal abnormalities are: t(l;14) (p22;q32); t(l4;l8)(q32; q21); t(3;l4)(q27;q32); t(6;l4)(p25,q32); t(lljl8)(q21;q21); t(ljl4)(q21; q32); 35 t(2j5)(p23;q35); add(l4q32) / dup(14p32); and

PB0004-W06-GB

t(11;14)(ql3;q32). Additional genetic loci, as yet undiscovered, are believed to account for other occurrences of NHL.

As another example, acute leukemia is a malignant 5 disease of bloodforming tissues such as the bone marrow.

It is characterized by the uncontrolled growth of white blood cells. As a result, immature myeloid cells (in acute myelogenous leukemia (AML)) or lymphoid cells (in acute lymphocytic leukemia (ALL)) rapidly accumulate and lo progressively replace the bone marrow; diminished production of normal red cells, white cells, and platelets ensues. This loss of normal marrow function in turn gives rise to the typical clinical complications of leukemia: anemia, infection, and bleeding.

15 If untreated, ALL is rapidly fatal; most patients die within several months of diagnosis. With appropriate therapy, many patients can be cured. The survival rate for patients diagnosed with AML or ALL is 14% and 58% respectively. However, the incidences of AML is expected 20 to be greater than ALL: an estimated 10,000 new cases of AML, predominantly in older adults, is anticipated in the U.S. alone, whereas 3,100 new cases of ALL are expected, with 1,500 of these new cases occurring among children.

The etiology of acute leukemia is not known.

25 Although human T-cell lymphotropic virus type I (HTLV-I), a causative agent of adult T-cell leukemia, and HTLV-II, obtained from several patients with a syndrome resembling hairy cell leukemia, have been isolated, the etiologic link between HTLV and malignancy is uncertain. There is, 30 however, evidence which suggests a genetic predisposition to incidences of acute leukemia.

For example, genetic disorders such as Fanconi anemia and Down syndrome appear to increase risk of acute leukemia, specifically, AML. Evidence supporting a 35 chromosome 21 locus for acute myelogenous leukemia (AML)

PB0004-WO6-GB

includes the finding of linkage to 21q22.1-q22.2 in a family with a platelet disorder and propensity to develop AML (Ho et al., Blood 87: 5218-5224 (1996), an increased incidence of leukemia in Down syndrome, and frequent 5 somatic translocation in leukemia involving the CBFA gene on 21q22.3. In addition, Horwitz et al., Am. J. Hum.

Genet. 61:873-881 (1997), suggest that a gene on 16q22 may be a second cause of acute myelogenous leukemia.

Nonparametric linkage analysis gave a P-value of 0.00098 10 for the conditional probability of linkage. Mutational analysis excluded expansion of the AT-rich minisatellite repeat FRA16B fragile site and the CAG trinucleotide repeat in the E2F-4 transcription factor. Large CAG repeat expansion was excluded as a cause of leukemia in this 15 family.

Similarly, acute lymphoblastic leukemia (ALL) has been suggested to have a genetic predisposition. In particular, linkage to chromosome 9p has been reported by a number of groups. Chilcote et al., New Eng. J. Med. 313: 20 286-291 (1985), found that 6 of 8 patients with clinical features of lymphomatous ALL (LALL), a distinct category of ALL of T-cell lineage, had karyotypic abnormalities leading to loss of bands 9p22-p21. The mechanisms varied and included deletions, unbalanced translocations, and loss of 25 the entire chromosome; only 1 of 57 patients without LALL had an abnormality of chromosome 9 at diagnosis. Kowalczyk et al., Cancer Genet. Cytogenet. 9:383-385 (1981), had earlier found changes in 9p in a subgroup of ALL cases.

Chilcote et al. (1985) pointed out that there is a fragile 30 site at 9p21 and raised the question of familial predisposition on this basis. This fragile site is the breakpoint in the translocation t(9;ll)(p21-22; q23), which is associated with acute nonlymphocytic leukemia with monocytic features, ANLL-AMoL-M5a. In a large series, 35 Murphy et al., New Eng. J. Med. 313:1611 (1985), confirmed

PB0004-W06-GB

an abnormality of 9p in 10 to 11% of cases (33 out of more than 300) of acute lymphoblastic leukemia. The breakpoints in 9p clustered in the p22p21 region. They could not, however, corroborate the specific association with T-cell 5 origin or so-called lymphomatous clinical features. In addition, Taki et al., Proc. Natl. Acad. Sci. USA 96:14535 (1999), recently identified AF5q31, a new AF4-related gene, fused to ALL in infant ALL with ins(5;ll)(q31;ql3q23), and suspects that AF5q31 and AF4 might define a new family 10 particularly involved in the pathogenesis of llq23-

associated-ALL. As yet a further example of a disease affecting bone marrow with likely polygenic etiology is multiple myeloma (MM).

15 MM is a cancer of plasma cells, the final differentiated stage of B lymphocyte maturation. The malignant clone proliferates in the bone marrow and frequently invades the adjacent bone, producing extensive skeletal destruction that results in bone pain and 20 fractures. Anemia, hypercalcemia, and renal failure are some clinical manifestations associated with MM.

MM causes 1% of all cancer deaths in Western countries. A genetic component to its etiology is suggested by disparate incidence among various groups in 2s the country. Its incidence is higher in men than in women, in people of African descent relative to the U.S. population at large, and in older adults as compared to the young. It has been estimated that 14,000 new cases of myeloma will be diagnosed in the U.S., and over 11,000 30 persons will die from MM within the year.

Although, Kaposi's sarcoma-associated herpes virus has been associated with MM (Retig et al., Science 276:1851 (1997)), there is evidence that chromosomal abnormalities, such as the deletion of 13ql4 and 35 rearrangements of 14q increase the proliferation of myeloma

PB0004-W06-GB

cells. Up to 30% of patients who suffer with MM have a balanced translocation, t(4;l4)(pl6.3;q32), that places the fibroblast growth factor receptor 3 (FGFR3) gene under the 5 control of IgH promoter elements (Chest et al., Nat. Genet.

16:260 (1997)). This results in increased expression of FGFR3, a member of a family of tyrosine kinase receptors implicated in control of cellular proliferation.

According to Zoger et al., Blood 95:1925 (2000), 10 monoallelic deletions of the retinoblastoma-1 (rb-1) gene and the D13S319 locus were observed in 48 of 104 patients (46.2%) and in 28 of 72 (38.9%) patients, respectively, with newly diagnosed MM. Fluorescence in situ-

hybridization (FISH) studies found that 13ql4 was deleted 15 in all 17 patients with karyotypic evidence of monosomy 13 or deletion of 13q but also in 9 of 19 patients with apparently normal karyotypes. Patients with a 13ql4 deletion were more likely to have higher serum levels of beta(2)microglobulin (P=0.059) and a higher percentage of 20 bone marrow plasma cells (P=0.085) than patients with a normal 13ql4 status on FISH analysis. In patients with a deletion of 13ql4, myeloma cell proliferation was markedly increased. The presence of a 13ql4 deletion on FISH analysis was associated with a significantly lower rate of 25 response to conventionaldose chemotherapy (40.8% compared with 78.6%; P =.009) and a shorter overall survival (24.2 months compared with > 60 months; P <.005) than in patients without the deletion.

There are numerous other mutated genes and 30 chromosomal abnormalities that may predispose to MM.

Examples of such genes are: B2M (15q21-q22); CCND1 (DllS287E, Cyclin D, PRADl)(llql3); CD19 (16pll.2); HGF (HPTA)(7q21.1); IL6 (IFNB2)(7p21); IRF4 (MUM1, LSIRF)(6p25-

p23); LTA (TNFB, LT)(6p21.3); SDC1 (2p24.1); and TNF (TNFA, 35 TNFSF2, DIF)(6p21.3). Examples of chromosomal

PB0004-W06-GB

abnormalities include: t(6;l4)(p25;q32) and t(lljl4)(ql3;q32). Other significant diseases or disorders of the bone marrow are also believed, or likely to have, a 5 genetic, typically polygenic, etiologic component. These diseases include, for example, chronic myeloid leukemia, chronic lymphoid leukemia, polycythemia vera, myelofibrosis, primary thrombocythemia, myelodysplastic syndromes, Wiskott-Aldrich, lymphoproliferative syndrome, 10 aplastic anemia, Fanconi anemia, Down syndrome, sickle cell disease, thalassemia, granulocyte disorders, Kostmann syndrome, chronic granulomatous disease, Chediak-Higashi syndrome, platelet disorders, Glanzmann thrombasthenia, Bernard-Soulier syndrome, metabolic storage diseases, 15 osteoporosis, congenital hemophagocytic syndrome.

The human genome-derived single exon nucleic acid probes and microarraysof the present invention are useful for predicting, diagnosing, grading, staging, monitoring and prognosing diseases of human bone marrow, particularly 20 those diseases with polygenic etiology. With each of the single exon probes described herein shown to be expressed at detectable levels in human bone marrow, and with about 2/3 of the probes identifying novel genes, the single exon microarrays of the present invention provide exceptionally 25 high informational content for such studies.

For example, diagnosis, grading, and/or staging of a disease can be based upon the quantitative relatedness of a patient gene expression profile to one or more reference expression profiles known to be characteristic of 30 a given bone marrow disease, or to specific grades or stages thereof.

In one embodiment, the patient gene expression profile is generated by hybridizing nucleic acids obtained directly or indirectly from transcripts expressed in the 3s patient's bone marrow (or cells cultured therefrom) to the

l PB0004-W06-GB

genome-derived single exon microarray of the present invention. Reference profiles are obtained similarly by hybridizing nucleic acids obtained directly or indirectly from transcripts expressed in the bone marrow of s individuals with known disease. Methods for quantitatively relating gene expression profiles, without regard to the function of the protein encoded by the gene, are disclosed in WO 99/58720, incorporated herein by reference in its entirety. 10 In another approach, the genome-derived single exon probes and microarrays of the present invention can be used to interrogate genomic DNA, rather than pools of expressed message; this latter approach permits predisposition to and/or prognosis of diseases of bone 15 marrow to be assessed through the massively parallel determination of altered copy number, deletion, or mutation in the patient's genome of exons known to be expressed in human bone marrow. The algorithms set forth in WO 99/58720 can be applied to such genomic profiles without regard to 20 the function of the protein encoded by the interrogated gene. The utility is specific to the probe; at sufficiently high hybridization stringency, which stringencies are well known in the art - see Ausubel et al. 25 and Maniatis et al. - each probe reports the level of expression of message specifically containing that ORF.

It should be appreciated, however, that the probes of the present invention, for which expression in the bone marrow has been demonstrated are useful for both 30 measurement in the bone marrow and for survey of expression in other tissues.

Significant among such advantages is the presence of probes for novel genes.

As mentioned above and further detailed in 35 Examples l and 2, the methods described enable ORFs which

PB0004-W06-GB

are not present in existing expression databases to be identified. And the fewer the number of tissues in which the ORF can be shown to be expressed, the more likely the ORF will prove to be part of a novel gene: as further 5 discussed in Example 2, ORFs whose expression was measurable in only a single of the tested tissues were represented in existing expression databases at a rate of only 11%, whereas 36% of ORFs whose expression was measurable in 9 tissues were present in existing expression 10 databases, and fully 45% of those ORFs expressed in all ten tested tissues were present in existing expressed sequence databases. Either as tools for measuring gene expression or tools for surveying gene expression, the genome-derived 15 single exon probes of the present invention have significant advantages over the cDNA or EST-based probes that are currently available for achieving these utilities.

The genome-derived single exon probes of the present invention are useful in constructing genome-derived 20 single exon microarrays; the genomederived single exon microarrays, in turn, are useful devices for measuring and for surveying gene expression in the human.

Gene expression analysis using microarrays -

conventionally using microarrays having probes derived from 25 expressed message - is well-established as useful in the biological research arts (see Lockhart et al. Nature 405, 827-836).

Microarrays have been used to determine gene expression profiles in cells in response to drug treatment 30 (see, for example, Kaminski et al., "Global Analysis of Gene Expression in Pulmonary Fibrosis Reveals Distinct Programs Regulating Lung Inflammation and Fibrosis," Proc. Natl. Acad. sci. USA 97(4):1778-83 (2000); Bartosiewicz et al., "Development of a Toxicological Gene Array and 35 Quantitative Assessment of This Technology," Arch. Biochem.

PB0004-W06-GB

: Biophys. 376(1):66-73 (2000)), viral infection (see for example, Geiss et al., ''Large-scale Monitoring of Host Cell Gene Expression During HIV1 Infection Using cDNA Microarrays," Virology 266(1):8-16 (2000)) and during cell 5 processes such as differentiation, senescence and apoptosis (see, for example, Shelton et al., "Microarray Analysis of Replicative Senescence," Curr. Biol. 9 (17):939-45 (1999); Voehringer et al., "Gene Microarray Identification of Redox and Mitochondrial Elements That Control Resistance or 10 Sensitivity to Apoptosis," Proc. Natl. Acad. Sci. USA 97(6):2680-5 (2000)).

Microarrays have also been used to determine abnormal gene expression in diseased tissues (see, for example, Alon et al., "Broad Patterns of Gene Expression 15 Revealed by Clustering Analysis of Tumor and Normal Colon Tissues Probed by Oligonucleotide Arrays," Proc. Natl. Acad. Sci. USA 96(12):6745-50 (1999); Perou et al., "Distinctive Gene Expression Patterns in Human Mammary Epithelial Cells and Breast Cancers, Proc. Natl. Acad. 5ci.

20 USA 96(16):9212-7 (1999); Wang et al., "Identification of Genes Differentially Over-expressed in Lung Squamous Cell Carcinoma Using Combination of cDNA Subtraction and Microarray Analysis," Oncogene 19 (12) :1519-28 (2000); Whitney et al., "Analysis of Gene Expression in Multiple 25 Sclerosis Lesions Using cDNA Microarrays," Ann. Neural.

46(3):425-8 (1999)), in drug discovery screens (see, for example, Scherf et al., "A Gene Expression Database for the Molecular Pharmacology of Cancer," Nat. Genet. 24(3):236-44 (2000)) and in diagnosis to determine appropriate treatment 30 strategies (see, for example, Sgroi et al., "In vivo Gene Expression Profile Analysis of Human Breast Cancer Progression, " Cancer Res. 59(22):5656-61 (1999)).

In microarray-based gene expression screens of pharmacological drug candidates upon cells, each probe

PB0004-W06-GB

provides specific useful data. In particular, it should be appreciated that even those probes that show no change in expression are as informative as those that do change, serving, in essence, as negative controls.

5 For example, where gene expression analysis is used to assess toxicity of chemical agents on cells, the failure of the agent to change a gene's expression level is evidence that the drug likely does not affect the pathway of which the gene's expressed protein is a part.

10 Analogously, where gene expression analysis is used to assess side effects of pharmacological agents - whether in lead compound discovery or in subsequent screening of lead compound derivatives - the inability of the agent to alter a gene's expression level is evidence that the drug does 15 not affect the pathway of which the gene's expressed protein is a part.

WO 99/58720 provides methods for quantifying the relatedness of a first and second gene expression profile and for ordering the relatedness of a plurality of gene 20 expression profiles. The methods so described permit useful information to be extracted from a greater percentage of the individual gene expression measurements from a microarray than methods previously used in the art.

Other uses of microarrays are described in 25 Gerhold et al., Trends Biochem. Sci. 24(5):168-173 (1999) and Zweiger, Trends Biotechnol. 17(11) :429-436 (1999); Schena et al. The invention particularly provides genome-

derived single-axon probes known to be expressed in bone 30 marrow. The individual single exon probes can be provided in the form of substantially isolated and purified nucleic acid, typically, but not necessarily, in a quantity sufficient to perform a hybridization reaction.

Such nucleic acid can be in any form directly 3s hybridizable to the message that contains the probe's ORF,

PB0004-W06-GB

such as double stranded DNA, single-stranded DNA complementary to the message, single-stranded RNA complementary to the message, or chimeric DNA/RNA molecules so hybridizable. The nucleic acid can alternatively or 5 additionally include either nonnative nucleotides, alternative internucleotide linkages, or both, so long as complementary binding can be obtained. For example, probes can include phosphorothioates, methylphosphonates, morpholino analogs, and peptide nucleic acids (PNA), as are 10 described, for example, in U.S. Patent Nos. 5,142,047; 5,235, 033; 5,166,315; 5,217,866; 5,184,444; 5,861,250.

Usefully, however, such probes are provided in a form and quantity suitable for amplification, where the amplified product is thereafter to be used in the 15 hybridization reactions that probe gene expression.

Typically, such probes are provided in a form and quantity suitable for amplification by PCR or by other well known amplification technique. One such technique additional to PCR is rolling circle amplification, as is described, inter 20 alla, in U.S. Patent Nos. 5,854,033 and 5,714,320 and international patent publications WO 97/19193 and WO 00/15779. As is well understood, where the probes are to be provided in a form suitable for amplification, the range of nucleic acid analogues and/or internucleotide 25 linkages will be constrained by the requirements and nature of the amplification enzyme.

Where the probe is to be provided in form suitable for amplification, the quantity need not be sufficient for direct hybridization for gene expression 30 analysis, and need be sufficient only to function as an amplification template, typically at least about 1, 10 or 100 pg or more.

Each discrete amplifiable probe can also be packaged with amplification primers, either in a single 35 composition that comprises probe template and primers, or

PB0004-W06-GB

in a kit that comprises such primers separately packaged therefrom. As earlier mentioned, the ORF-specific 5' primers used for genomic amplification can have a first common sequence added thereto, and the ORFspecific 3' 5 primers used for genomic amplification can have a second, different, common sequence added thereto, thus permitting, in this embodiment, the use of a single set of 5' and 3' primers to amplify any one of the probes. The probe composition and/or kit can also include buffers, enzyme, 10 etc., required to effect amplification.

As mentioned earlier, when intended for use on a genome-derived single exon microarray of the present invention, the genome-derived single exon probes of the present invention will typically average at least about 15 100, 200, 300, 400 or 500 bp in length, including (and typically, but not necessarily centered about) the ORF.

Furthermore, when intended for use on a genome-derived single exon microarray of the present invention, the genome-derived single exon probes of the present invention 20 will typically not contain a detectable label.

When intended for use in solution phase hybridization, however - that is, for use in a hybridization reaction in which the probe is not first bound to a support substrate (although the target may 25 indeed be so bound) length constraints that are imposed in microarray-based hybridization approaches will be relaxed, and-such probes will typically be labeled.

In such case, the only functional constraint that dictates the minimum size of such probe is that each such 30 probe must be capable of specifically identifying in a hybridization reaction the exon from which it is drawn. In theory, a probe of as little as 17 nucleotides is capable of uniquely identifying its cognate sequence in the human genome. For hybridization to expressed message - a subset 35 of target sequence that is much reduced in complexity as

PB0004-W06-GB

compared to genomic sequence - even fewer nucleotides are required for specificity.

Therefore, the probes of the present invention can include as few as 20, 25 or 50 bp or ORF, or more. In 5 particular embodiments, the ORF sequences are given in SEQ ID NOS. 13,115 - 26,012, respectively, for probe SEQ ID NOS. 1 - 13,114. The minimum amount of ORF required to be included in the probe of the present invention in order to provide specific signal in either solution phase or 10 microarray-based hybridizations can readily be determined for each of ORF SEQ ID NOS. 13, 115 - 26,012 individually by routine experimentation using standard high stringency conditions. Such high stringency conditions are described, 15 inter alla, in Ausubel et al. and Maniatis et al. For microarray-based hybridization, standard high stringency conditions can usefully be 50% formamide, 5X SSC, 0.2 1lg/p poly(dA), 0.2 g/'Ll human cotl DNA, and 0.5 % SDS, in a humid oven at 42 C overnight, followed by successive washes 20 of the microarray in 1X SSC, 0. 2% SDS at 55 C for 5 minutes, and then 0.1X SSC, 0.2% SDS, at 55 C for 20 minutes. For solution phase hybridization, standard high stringency conditions can usefully be aqueous hybridization at 65 C in 6X SSC. Lower stringency conditions, suitable 25 for cross-hybridization to mRNA encoding structurally- and functionally-related proteins, can usefully be the same as the high stringency conditions but with reduction in temperature for hybridization and washing to room temperature (approximately 25 C).

30 When intended for use in solution phase hybridization, the maximum size of the single exon probes of the present invention is dictated by the proximity of other expressed exons in genomic DNA: although each single exon probe can include intergenic and/or intronic material 35 contiguous to the ORF in the human genome, each probe of

PB0004-W06-GB

the present invention will include portions of only one expressed exon.

Thus, each single exon probe will include no more than about 25 kb of contiguous genomic sequence, more 5 typically no more than about 20 kb of contiguous genomic sequence, more usually no more than about 15 kb, even more usually no more than about 10 kb. Usually, probes that are maximally about 5 kb will be used, more typically no more than about 3 kb.

10 It will be appreciated that the Sequence Listing appended hereto presents, by convention, only that strand of the probe and ORF sequence that can be directly translated reading from 5' to 3' end. As would be well understood by one of skill in the art, single stranded 15 probes must be complementary in sequence to the ORF as present in an mRNA; it is well within the skill in the art to determine such complementary sequence. It will further be understood that double stranded probes can be used in both solution-phase hybridization and microarray-based 20 hybridization if suitably denatured.

Thus, it is an aspect of the present invention to provide single-stranded nucleic acid probes that have sequence complementary to those described herein above and below, and double-stranded probes one strand of which has 25 sequence complementary to the probes described herein.

The probes can, but need not, contain intergenic and/or intronic material that flanks the ORF, on one or both sides, in the same linear relationship to the ORF that the intergenic and/or intronic material bears to the ORF in 30 genomic DNA. The probes do not, however, contain nucleic acid derived from more than one expressed ORF.

And when intended for use in solution hybridization, the probes of the present invention can usefully have detectable labels. Nucleic acid labels are 3s well known in the art, and include, inter alia, radioactive

PB0004-WO6-GB

labels such as 3H 32p 33p 35S l25I l3lI; fluorescent labels, such as Cy3, Cy5, Cy5.5, Cy7, SYBR Green and other labels described in Haugland, Handbook of Fluorescent Probes and Research Chemicals, 7th 5 ea., Molecular Probes Inc., Eugene, OR (2000), or fluorescence resonance energy transfer tandem conjugates thereof; labels suitable for chemiluminescent and/or enhanced chemiluminescent detection; labels suitable for ESR and NMR detection; and labels that include one member 10 of a specific binding pair, such as biotin, digoxigenin, or the like.

The probes, either in quantity sufficient for hybridization or sufficient for amplification, can be provided in individual vials or containers.

15 Alternatively, such probes can usefully be packaged as a plurality of such individual genome-derived single exon probes.

When provided as a collection of plural individual probes, the probes are typically made available 20 in amplifiable form in a spatiallyaddressable ordered set, typically one per well of a microtiter dish. Although a 96 well microtiter plate can be used, greater efficiency is obtained using higher density arrays.

If, as earlier mentioned, the ORF-specific 25 5' primers used for genomic amplification had a first common sequence added thereto, and the ORFspecific 3' primers used for genomic amplification had a second, different, common sequence added thereto, a single set of 5' and 3' primers can be used to amplify all of the probes 30 from the amplifiable ordered set.

Such collections of genome-derived single exon probes can usefully include a plurality of probes chosen for the common attribute of expression in the human bone marrow. 3s In such defined subsets, typically at least 50,

PB0004-W06-GB

60, 75, 80, 85, 90 or 95% or more of the probes will be chosen by their expression in the defined tissue or cell type. The single exon probes of the present invention, 5 as well as fragments of the single exon probes comprising selectively hybridizable portions of the probe ORF, can be used to obtain the full length cDNA that includes the ORF by (i) screening of cDNA libraries; (ii) rapid amplification of c DNA ends ("RACE"); or (iii) other 10 conventional means, as are described, inter alla, in Ausubel et al. and Maniatis et al. It is another aspect of the present invention to provide genome-derived single exon nucleic acid microarrays useful for gene expression analysis, where the term 15 "microarray" has the meaning given in the definitional section of this description, supra.

The invention particularly provides genome-

derived single-axon nucleic acid microarrays comprising a plurality of probes known to be expressed in human bone 20 marrow. In preferred embodiments, the present invention provides human genome-derived single exon microarrays comprising a plurality of probes drawn from the group consisting of SEQ ID NOS.: 1 - 13,114.

When used for gene expression analysis, the 25 genome-derived single exon microarrays provide greater physical informational density than do the genome-derived single exon microarrays that have lower percentages of probes known to be expressed commonly in the tested tissue.

At a fixed probe density, for example, a given microarray 30 surface area of the defined subset genome-derived single exon microarray can yield a greater number of expression measurements. Alternatively, at a given probe density, the same number of expression measurements can be obtained from a smaller substrate surface area. Alternatively, at a 35 fixed probe density and fixed surface area, probes can be

PB0004-WO6-GB

provided redundantly, providing greater reliability in signal measurement for any given probe. Furthermore, with a higher percentage of probes known to be expressed in the assayed tissue, the dynamic range of the detection means s can be adjusted to reveal finer levels discrimination among the levels of expression.

Although particularly described with respect to their utility as probes of gene expression, particularly as probes to be included on a genomederived single exon 10 microarray, each of the nucleic acids having SEQ ID NOS.: 1 - 13,114 contains an open-reading frame, set forth respectively in SEQ ID NOS.: 13,115 - 26,012, that encodes a protein domain. Thus, each of SEQ ID NOS. 1 - 13,114 can be used, or that portion thereof in SEQ ID NOS. 13,115 15 26,012 used, to express a protein domain by standard in vitro recombinant techniques. See Ausubel et al. and Maniatis et al. Additionally, kits are available commercially that readily permit such nucleic acids to be expressed as 20 protein in bacterial cells, insect cells, or mammalian cells, as desired (e.g., HAT Protein Expression & Purification System, ClonTech Laboratories, Palo Alto, CA; Adeno-XTU Expression System, ClonTech Laboratories, Palo Alto, CA; Protein Fusion & Purification (pMALTU) System, New 25 England Biolabs, Beverley, MA) Furthermore, shorter peptides can be chemically synthesized using commercial peptide synthesizing equipment and well known techniques. Procedures are described, inter alla, in Chan et al. (eds.), Fmoc Solid Phase Peptide 30 Synthesis: A Practical Approach (Practical Approach Series, (Paper)), Oxford Univ. Press (March 2000) (ISBN: 0199637245); Jones, Amino Acid and Peptide Synthesis (Oxford Chemistry Primers, No 7), Oxford Univ. Press (August 1992) (ISBN: 0198556683); and Bodanszky, Principles 35 of Peptide Synthesis (Springer Laboratory), Springer Verlag

PB0004-W06-GB

*(December 1993) (ISBN: 0387564314).

It is, therefore, another aspect of the invention to provide peptides comprising an amino acid sequence translated from SEQ ID NOS.: 13,115 26,012. Such amino 5 acid sequences are set out in SEQ ID NOS: 26,013 38,628.

Any such recombinantly-expressed or synthesized peptide of at least 8, and preferably at least about 15, amino acids, can be conjugated to a carrier protein and used to generate antibody that recognizes the peptide. Thus, it is a 10 further aspect of the invention to provide peptides that have at least 8, preferably at least 15, consecutive amino acids. The following examples are offered by way of 15 illustration and not by way of limitation.

EXAMPLE 1

Preparation of Single Exon Microarrays from ORFs Predicted in Human Genomic Sequence Bioinformatics Results All human BAC sequences in fewer than 10 pieces that had been accessioned in a five month period immediately preceding this study were downloaded from 25 GenBank. This corresponds to 2200 clones, totaling 350 MB of sequence, or approximately 10% of the human genome.

After masking repetitive elements using the program CROSS_MATCH, the sequence was analyzed for open reading frames using three separate gene finding programs.

30 The three programs predict genes using independent algorithmic methods developed on independent training sets: GRAIL uses a neural network, GENEFINDER uses a hidden Markoff model, and DICTION, a program proprietary to Genetics Institute, operates according to a different 35 heuristic. The results of all three programs were used to

PB0004-W06-GB

create a prediction matrix across the segment of genomic DNA. The three gene finding programs yielded a range of results. GRAIL identified the greatest percentage of 5 genomic sequence as putative coding region, 2% of the data analyzed. GENEFINDER was second, calling 1%, and DICTION yielded the least putative coding region, with 0.8% of genomic sequence called as coding region.

The consensus data were as follows. GRAIL and 10 GENEFINDER agreed on 0. 7% of genomic sequence, GRAIL and DICTION agreed on 0.5% of genomic sequence, and the three programs together agreed on 0.25% of the data analyzed.

That is, 0.25% of the genomic sequence was identified by all three of the programs as containing putative coding 15 region.

ORFs predicted by any two of the three programs ("consensus ORFs") were assorted into "gene bins" using two criteria: (1) any 7 consecutive exons within a 25 kb window were placed together in a bin as likely contributing to a 20 single gene, and (2) all ORFs within a 25 kb window were placed together in a bin as likely contributing to a single gene if fewer than 7 exons were found within the 25 kb window. 25 PCR

The largest ORF from each gene bin that did not span repetitive sequence was then chosen for amplification, as were all consensus ORFs longer than 500 bp. This method approximated one exon per gene; however, a number of genes 30 were found to be represented by multiple elements.

Previously, we had determined that DNA fragments fewer than 250 bp in length do not bind well to the amino-

modified glass surface of the slides used as support substrate for construction of microarrays; therefore, 35 amplicons were designed in the present experiments to

PB0004-W06-GB

approximate 500 bp in length.

Accordingly, after selecting the largest ORF per gene bin, a 500 bp fragment of sequence centered on the ORF was passed to the primer picking software, PRIMERS 5 (available online for use at http://www-genome.wi.mit. edu/cgi-bin/primer/). A first additional sequence was commonly added to each ORF-unique 5' primer, and a second, different, additional sequence was commonly added to each ORF-unique 3' primer, to permit 10 subsequent reamplification of the amplicon using a single set of "universal" 5' and 3' primers, thus immortalizing the amplicon. The addition of universal priming sequences also facilitates sequence verification, and can be used to add a cloning site should some ORFs be found to warrant 15 further study.

The ORFs were then PCR amplified from genomic DNA, verified on agarose gels, and sequenced using the universal primers to validate the identity of the amplicon to be spotted in the microarray.

20 Primers were supplied by Operon Technologies (Alameda, CA). PCR amplification was performed by standard techniques using human genomic DNA (Clontech, Palo Alto, CA) as template. Each PCR product was verified by SYBR green (Molecular Probes, Inc., Eugene, OR) staining of 25 agarose gels, with subsequent imaging by Fluorimager (Molecular Dynamics, Inc., Sunnyvale, CA). PCR amplification was classified as successful if a single band appeared. The success rate for amplifying ORFs of interest 30 directly from genomic DNA using PCR was approximately 75%.

FIG. 5 graphs the distribution of predicted ORF (exon) length and distribution of amplified PCR products, with ORF length shown in red and PCR product length shown in blue (which may appear black in the figure). Although the range 35 of ORF sizes is readily seen to extend to beyond 900 bp,

PB0004-W06-GB

the mean predicted exon size was only 229 bp, with a median size of 150 bp (n=9498). With an average amplicon size of 475 + 25 bp, approximately 50% of the average PCR amplification product contained predicted coding region, 5 with the remaining 50% of the amplicon containing either intron, intergenic sequence, or both.

Using a strategy predicated on amplifying about 500 bp, it was found that long exons had a higher PCR failure rate. To address this, the bioinformatics process lo was adjusted to amplify 1000, 1500 or 2000 bp fragments from exons larger than 500 bp. This improved the rate of successful amplification of exons exceeding 500 bp, constituting about 9. 2% of the exons predicted by the gene finding algorithms.

15 Approximately 75% of the probes disposed on the array (90% of those that successfully PCR amplified) were sequence-verified by sequencing in both the forward and reverse direction using MegaBACE sequencer (Molecular Dynamics, Inc., Sunnyvale, CA), universal primers, and 20 standard protocols.

Some genomic clones (BACs) yielded very poor PCR and sequencing results. The reasons for this are unclear, but may be related to the quality of early draft sequence or the inclusion of vector and host contamination in some 25 submitted sequence data.

Although the intronic and intergenic material flanking coding regions could theoretically interfere with hybridization during microarrayexperiments, subsequent empirical results demonstrated that differential expression 30 ratios were not significantly affected by the presence of noncoding sequence. The variation in exon size was similarly found not to affect differential expression ratios significantly; however, variation in exon size was observed to affect the absolute signal intensity (data not 35 shown).

PB0004-W06-GB

The 350 MB of genomic DNA was, by the above-

described process, reduced to 9750 discrete probes, which were spotted in duplicate onto glass slides using commercially available instrumentation (MicroArray GenII 5 Spotter and/or MicroArray GenIII Spotter, Molecular Dynamics, Inc., Sunnyvale, CA). Each slide additionally included either 16 or 32 E. cold genes, the average hybridization signal of which was used as a measure of background biological noise.

10 Each of the probe sequences was BLASTed against the human EST data set, the NR data set, and SwissProt GenBank (May 7, 1999 release 2.0.9).

One third of the probe sequences (as amplified) produced an exact match (BLAST Expect ("E") values less 15 than 1 el ) to either an EST (20% of sequences) or a known mRNA (13% of sequences). A further 22% of the probe sequences showed some homology to a known EST or mRNA (BLAST E values from 1 en to 1 e99). The remaining 45% of the probe sequences showed no significant sequence homology 20 to any expressed, or potentially expressed, sequences present in public databases.

All of the probe sequences (as amplified) were then analyzed for protein similarities with the SwissProt database using BLASTX, Gish et al., Nature Genet. 3:266 25 (1993). The predicted functional breakdowns of the 2/3 of probes identical or homologous to known sequences are presented in Table 1.

Table 1

Function of Predicted ORFs As Deduced From Comparative Sequence Analysis Total V6 chip V7 chip Function Predicted from Comparative Sequence

PB0004-W06-GB

Analysis 211 115 Receptor 120 43 77 Zinc Finger O 11 19 Homeobox 25 16 Transcription Factor 17 11 7 Transcription 118 57 61 Structural 95 39 56 Kinase 36 18 18 Phosphatase 83 31 52 Ribosomal 45 19 26 Transport 21 17 14 Growth Factor 17 12 Cytochrome 50 33 17 Channel As can be seen, the two most common types of genes were transcription factors and receptors, making up 2.2% and 1.8% of the arrayed elements, respectively.

EXAMPLE 2

Gene Expression Measurements From Genome-Derived Single Exon Microarrays The two genome-derived single exon microarrays prepared according to Example 1 were hybridized in a series of simultaneous two-color fluorescence experiments to (1) 15 Cy3-labeled cDNA synthesized from message drawn individually from each of brain, heart, liver, fetal liver, placenta, lung, bone marrow, HeLa, BT 474, or HBL 100 cells, and (2) Cy5labeled cDNA prepared from message pooled from all ten tissues and cell types, as a control in 20 each of the measurements. Hybridization and scanning were

PB0004-WO6-GB

carried out using standard protocols and Molecular Dynamics equipment. Briefly, mRNA samples were bought from commercial sources (Clontech, Palo Alto, CA and Amersham Pharmacia 5 Biotech (APB)). Cy3-dCTP and Cy5-dCTP (both from APB) were incorporated during separate reverse transcriptions of 1 fig of polyA+ mRNA performed using 1 fig oligo(dT)12-18 primer and 2, ug random 9mer primers as follows. After heating to 70 C, the RNA:primer mixture was snap cooled on ice. After 10 snap cooling on ice, added to the RNA to the stated final concentration was: 1X Superscript II buffer, 0.01 M DTT, lOO,uM dATP, 100 AM dGTP, 100 EM dTTP, 50 AM dCTP, 50 AM Cy3- dCTP or Cy5-dCTP 50 I1M, and 200 U Superscript II enzyme. The reaction was incubated for 2 hours at 42 C.

15 After 2 hours, the first strand cDNA was isolated by adding 1 U Ribonuclease H. and incubating for 30 minutes at 37 C.

The reaction was then purified using a Qiagen PCR cleanup column, increasing the number of ethanol washes to 5.

Probe was eluted using 10 mM Tris pH 8.5.

20 Using a spectrophotometer, probes were measured for dye incorporation. Volumes of both Cy3 and Cy5 cDNA corresponding to 50 pmoles of each dye were then dried in a Speedvac, resuspended in 30 Ill hybridization solution containing 50% formamide, 5X SSC, 0.2 g/lll poly(dA), 0.2 25 1lg/l human cotl DNA, and 0.5 % SDS.

Hybridizations were carried out under a coverslip, with the array placed in a humid oven at 42 C overnight. Before scanning, slides were washed in 1X SSC, 0.2% SDS at 55 C for 5 minutes, followed by O.1X SSC, 0.2% 30 SDS, at 55 C for 20 minutes. Slides were briefly dipped in water and dried thoroughly under a gentle stream of nitrogen. Slides were scanned using a Molecular Dynamics Gen3 scanner, as described. Schena (ed.), Microarray 35 Biochip: Tools and Technology, Eaton Publishing

PB0004-W06-GB

Company/BioTechniques Books Division (2000) (ISBN: 1881299376).

Although the use of pooled cDNA as a reference permitted the survey of a large number of tissues, it 5 attenuates the measurement of relative gene expression, since every highly expressed gene in the tissue/cell type-

specific fluorescence channel will be present to a level of at least 10% in the control channel. Because of this fact, both signal and expression ratios (the latter hereinafter, 10 "expression" or "relative expression") for each probe were normalized using the average ratio or average signal, respectively, as measured across the whole slide.

Data were accepted for further analysis only when signal was at least three times greater than biological 15 noise, the latter defined by the average signal produced by the E. cold control genes.

The relative expression signal for these probes was then plotted as function of tissue or cell type, and is presented in FIG. 6.

20 FIG. 6 shows the distribution of expression across a panel of ten tissues. The graph shows the number of sequence-verified products that were either not expressed ("0"), expressed in one or more but not all tested tissues ("1" - "9"), and expressed in all tissues 25 tested ("10").

Of 9999 arrayed elements on the two microarrays (including positive and negative controls and "failed" products), 2353 (51%) were expressed in at least one tissue or cell type. Of the gene elements showing significant 30 signal - where expression was scored as "significant" if the normalized Cy3 signal was greater than 1, representing signal 5-fold over biological noise (0.2) - 39% (991) were expressed in all 10 tissues. The next most common class (15%) consisted of gene elements expressed in only a single 35 tissue.

PB0004-W06-GB

The genes expressed in a single tissue were further analyzed, and the results of the analyses are compiled in FIG. 7.

FIG. 7A is a matrix presenting the expression of 5 all verified sequences that showed expression greater than 3 in at least one tissue. Each clone is represented by a column in the matrix. Each of the 10 tissues assayed is represented by a separate row in the matrix, and relative expression of a clone in that tissue is indicated at the 10 respective node by intensity of green shading, with the intensity legend shown in panel B. The top row of the matrix ("EST Hit") contains "bioinformatic" rather than "physical" expression data - that is, presents the results returned by query of EST, NR and SwissProt databases using 15 the probe sequence. The legend for "bioinformatic expression" (i.e., degree of homology returned) is presented in panel C. Briefly, white is known, black is novel, with gray depicting nonidentical with significant homology (white: E values c le-100; gray: E values from le 20 05 to le-99; black: E values > le-05).

As FIG. 7 readily shows, heart and brain were demonstrated to have the greatest numbers of genes that were shown to be uniquely expressed in the respective tissue. In brain, 200 uniquely expressed genes were 25 identified; in heart, 150. The remaining tissues gave the following figures for uniquely expressed genes: liver, 100; lung, 70; fetal liver, 150; bone marrow, 75; placenta, 100; HeLa, 50; HBL, 100; and BT474, 50.

It was further observed that there were many more 30 "novel" genes among those that were up-regulated in only one tissue, as compared with those that were down-regulated in only one tissue. In fact, it was found that ORFs whose expression was measurable in only a single of the tested tissues were represented in sequencing databases at a rate 35 of only 11%, whereas 36% of the ORFs whose expression was

PB0004-W06-GB

measurable in 9 of the tissues were present in public databases. As for those ORFs expressed in all ten tissues, fully 45% were present in existing expressed sequence databases. These results are not unexpected, since genes 5 expressed in a greater number of tissues have a higher likelihood of being, and thus of having been, discovered by EST approaches.

Comparison of Signal from Known and Unknown Genes 10 The normalized signal of the genes found to have high homology to genes present in the GenBank human EST database were compared to the normalized signal of those genes not found in the GenBank human EST database. The data are shown in FIG. 8.

15 FIG. 8 shows the normalized Cy3 signal intensity for all sequenceverified products with a BLAST Expect ("E") value of greater than le-30 (designated "unknown") upon query of existing EST, NR and SwissProt databases, and shows in blue the normalized Cy3 signal intensity for all 20 sequence-verified products with a BLAST Expect value of less than le30 ("known"). Note that biological background

noise has an averaged normalized Cy3 signal intensity of 0.2. As expected, the most highly expressed of the 25 ORFs were "known" genes. This is not surprising, since very high signal intensity correlates with very commonly-

expressed genes, which have a higher likelihood of being found by EST sequence.

However, a significant point is that a large 30 number of even the high expressers were "unknown". Since the genomic approach used to identify genes and to confirm their expression does not bias exons toward either the 3' or 5' end of a gene, many of these high expression genes will not have been detected in an end-sequenced cDNA 35 library.

PB0004-W06-GB

The significant point is that presence of the gene in an EST database is not a prerequisite for incorporation into a genome-derived microarray, and further, that arraying such "unknown" exons can help to 5 assign function to as-yet undiscovered genes.

Verification of Gene Expression To ascertain the validity of the approach described above to identify genes from raw genomic 10 sequence, expression of two of the probes was assayed using reverse transcriptase polymerase chain reaction (RT PCR) and northern blot analysis.

Two microarray probes were selected on the basis of exon size, prior sequencing success, and tissue-specific 15 gene expression patterns as measured by the microarray experiments. The primers originally used to amplify the two respective ORES from genomic DNA were used in RT PCR against a panel of tissue-specific cDNAs (Rapid-Scan gene expression panel 24 human cDNAs) (OriGene Technologies, 20 Inc., Rockville, MD).

Sequence AL079300_1 was shown by microarray hybridization to be present in cardiac tissue, and sequence AL031734_1 was shown by microarray experiment to be present in placental tissue (data not shown). RT-PCR on these two 25 sequences confirmed the tissue-specific gene expression as measured by microarrays, as ascertained by the presence of a correctly sized PCR product from the respective tissue type cDNAs.

Clearly, all microarray results cannot, and 30 indeed should not, be confirmed by independent assay methods, or the high throughput, highly parallel advantages of microarray hybridization assays will be lost. However, in addition to the two RT-PCR results presented above, the observation that 1/3 of the arrayed genes exist in 35 expression databases provides powerful confirmation of the

PB0004-W06-GB

power of our methodology - which combines bioinformatic prediction with expression confirmation using genome derived single exon microarrays - to identify novel genes from raw genomic data.

5 To verify that the approach further provides correct characterization of the expression patterns of the identified genes, a detailed analysis was performed of the microarrayed sequences that showed high signal in brain.

For this latter analysis, sequences that showed 10 high (normalized) signal in brain, but which showed very low (normalized) signal (less than 0.5, determined to be biological noise) in all other tissues, were further studied. There were 82 sequences that fit these criteria, approximately 2% of the arrayed elements. The 10 sequences 15 showing the highest signal in brain in microarray hybridizations are detailed in Table 2, along with assigned function, if known or reasonably predicted.

Table 2

Function of the Most Highly Expressed Genes Expressed Only in Brain Microarray Normal Expressi Homology Gene Function Sequence ized on Ratio to EST as described by Name Signal present GenBank in GenBank AP000217- 1 5.2 +7.7 High S-100 protein, lo-chain, Ca2+ binding protein expressed in central nervous AP000047-1 2.3 High Unknown

PB0004-W06-GB

Function AC006548-9 1.7 High Similar to mouse membrane glyco-protein Me, expressed in central nervous system AC007245-5 1.5 High Similar to amphiphysin, a synaptic vesicle associated protein. Ref 21 L44140-4 1.2 + 2.0 High Endothelial act in-binding protein found in nonmuscle filamin AC004689-9 1.2 +3.5 High Protein Phosphatase PP2A, neuronal/ downregulates activated protein kineses AL031657-1 1.2 +3.0 High Unknown function/ Contains the anhyrin motif, a common protein sequence motif AC009266-2 1.1 +3.7 Low Low homology to Synaptotagmin I

PB0004-W06 -GB

protein in rat/present at low levels throughout rat brain AP000086-1 1.0 + 2.7 Low Unknown, very poor homology to collagen AC004689-3 1. 0 High Protein Phosphatase PP2A, neuronal/ downregul at es activated protein kineses Of the ten sequences studied by these latter confirmatory approaches, eight were previously known. Of these eight, six had previously been reported to be 5 important in the central nervous system or brain. The exon giving the highest signal (AP00217-1) was found to be the gene encoding an STOOP Ca2+ binding protein, reported in the literature to be highly and uniquely expressed in the central nervous system. Heizmann, Neurochem. Res. 9:1097 10 (1997).

A number of the brain-specific probe sequences (including AC006548-9, AC009266-2) did not have homology to any known human cDNAs in GenBank but did show homology to rat and mouse cDNAs. Sequences AC004689-9 and AC004689-3 15 were both found to be phosphatases present in neurons (Millward et al., Trends Biochem. Sci. 24(5):186-191 (1999)). Two microarray sequences, AP000047-1 and AP000086-1 have unknown function, with AP000086-1 being absent from GenBank. Functionality can now be narrowed 20 down to a role in the central nervous system for both of these genes, showing the power of designing microarrays in

PB0004-WO6-GB

this fashion.

Next, the function of the chip sequences with the highest (normalized) signal intensity in brain, regardless of expression in other tissues, was assessed. In this 5 latter analysis, we found expression of many more common genes, since the sequences were not limited to those expressed only in brain. For example, looking at the 20 highest signal intensity spots in brain, 4 were similar to tubulin (AC00807905; AF146191-2; AC007664-4; AF14191-2), 2 10 were similar to actin (AL035701-2; AL034402- 1), and 6 were found to be homologous to glyceraldehyde-3-phosphate debydrogenase (GAPDH) (AL035604-1; Z86090-1; AC006064-L, AC006064-K; AC035604-3; AC006064-L). These genes are often used as controls or housekeeping genes in microarray 15 experiments of all types.

Other interesting genes highly expressed in brain were a ferritin heavy chain protein, which is reported in the literature to be found in brain and liver (Josh) et al., A. Neurol. Sci. 134(Suppl):52-56 (1995)), a result 20 duplicated with the array. Other highly expressed chip sequences included a translation elongation factor TO (AC007564-4), a DEAD-box homolog (AL023804-4), and a Y chromosome RNA-binding motif (Chai et al., Genomics 49(2):283-89 (1998))(AC007320-3). A low homology analog 25 (AP00123-l/2) to a gene, DSCR1, thought to be involved in trisomy 21 (Down's syndrome), showed high expression in both brain and heart, in agreement with the literature (Fuentes et al., Mol. Genet. 4(10):1935-44 (1995)).

As a further validation of the approach, we 30 selected the BAC AC006064 to be included on the array.

This BAC was known to contain the GAPDH gene, and thus could be used as a control for the ORF selection process.

The gene finding and exon selection algorithms resulted in choosing 25 exons from BAC AC006064 for spotting onto the 35 array, of which four were drawn from the GAPDH gene. Table

PB0004-W06-GB

3 shows the comparison of the average expression ratio for the 4 exons from BAC006064 compared with the average expression ratio for 5 different dilutions of a commercially available GAPDH cDNA (Clontech).

Table 3

Comparison of Expression Ratio, for each tissue, of GAPDH |ACOO6064 (n = 4) 1 Control ( n = 5) Bone Marrow -1.81 + 0.11 -1.85 + 0.08 Brain -1.41 + 0.11 -1.17 + 0.05 BT474 1.85 + 0.09 1.66 + 0.12

Fetal Liver -1.62 + 0.07 -1.41 + 0.05 HBL100 1.32 + 0.05 2.64 + 0.12

Heart 1.16 + 0.09 1.56 + 0.10 HeLa 1.11 +0.06 1.30 + 0.15 Liver -1.62 + 0. 22 -2.07 + Lung -4.95 + 0.93 -3.75 + 0.21 Placenta -3.56 + 0.25 -3.52 + 0. 43 Each tissue shows excellent agreement between the 10 experimentally chosen exons and the control, again demonstrating the validity of the present exon mining approach. In addition, the data also show the variability of expression of GAPDH within tissues, calling into question its classification as a housekeeping gene and 15 utility as a housekeeping control in microarray experiments. EXAMPLE 3

Representation of Sequence and Expression Data as a 20 "Mondrian"

PB0004-W06-GB

For each genomic clone processed for microarray as above-described, a plethora of information was accumulated, including full clone sequence, probe sequence 5 within the clone, results of each of the three gene finding programs, EST information associated with the probe sequences, and microarray signal and expression for multiple tissues, challenging our ability to display the information. 10 Accordingly, we devised a new tool for visual display of the sequence with its attendant annotation which, in deference to its visual similarity to the paintings of Piet Mondrian, is hereinafter termed a "Mondrian". FIGS. 3 and 4 present the key to the 15 information presented on a Mondrian.

FIG. 9 presents a Mondrian of BAC AC008172 (bases 25,000 to 130,000 shown) , containing the carbamyl phosphate synthetase gene (AF154830.1). Purple background within the

region shown as field 81 in FIG. 3 indicates all 37 known

20 exons for this gene.

As can be seen, GRAIL II successfully identified 27 of the known exons (73%), GENEFINDER successfully identified 37 of the known exons (100%), while DICTION identified 7 of the known exons (19%).

25 Seven of the predicted exons were selected for physical assay, of which 5 successfully amplified by PCR and were sequenced. These five exons were all found to be from the same gene, the carbamyl phosphate synthetase gene (AF154830.1).

30 The five exons were arrayed, and gene expression measured across 10 tissues. As is readily seen in the Mondrian, the five chip sequences on the array show identical expression patterns, elegantly demonstrating the reproducibility of the system.

35 FIG. 10 is a Mondrian of BAC AL049839. We

PB0004-W06-GB

selected 12 exons from this BAC, of which 10 successfully sequenced, which were found to form between 5 and 6 genes.

Interestingly, 4 of the genes on this BAC are protease inhibitors. Again, these data elegantly show that exons 5 selected from the same gene show the same expression patterns, depicted below the red line. From this figure, it is clear that our ability to find known genes is very good. A novel gene is also found from 86.6 kb to 88.6 kb, upon which all the exon finding programs agree. We are lo confident we have two exons from a single gene since they show the same expression patterns and the exons are proximal to each other. Backgrounds in the following

colors indicate a known gene (top to bottom): red = kallistatin protease inhibitor (P29622); 15 purple = plasma serine protease inhibitor (P05154); turquoise = al anti-chymotrypsin (P01011); mauve = 40S ribosomal protein (P08865). Note that chip sequence 8 and 12 did not sequence verify.

EXAMPLE 4

Genome-Derived Single Exon Probes Useful For Measuring Human Gene Expression 25 The protocols set forth in Examples 1 and 2, supra, were applied to additional human genomic sequence as it became newly available in GenBank to identify unique exons in the human genome that could be shown to be expressed at significant levels in bone marrow tissue.

30 These unique exons are within longer probe sequences. Each probe was completely sequenced on both strands prior to its use on a genome-derived single exon microarray; sequencing confirms the exact chemical structure of each probe. An added benefit of sequencing is 35 that it placed us in possession of a set of single base

PB0004-W06-GB

incremented fragments of the sequenced nucleic acid, starting from the sequencing primer 3' OH. (Since the single exon probes were first obtained by PCR amplification from genomic DNA, we were of course additionally in 5 possession of an even larger set of single base incremented fragments of each of the 13,114 single exon probes, each fragment corresponding to an extension product from one of the two amplification primers.) The structures of the 13,114 unique single exon 10 probes are clearly presented in the Sequence Listing as SEQ ID Nos.: 1 - 13,114. The 16 nt 5' primer sequence and 16 nt 3' primer sequence present on the amplicon are not included in the sequence listing. The sequences of the exons present within each of these probes is presented in 15 the Sequence Listing as SEQ ID Nos.: 13,115 - 26,012, respectively. It will be noted that some amplicons have more than one exon, some exons are contained in more than one amplicon.

AS detailed in Example 2, expression was 20 demonstrated by disposing the amplicons as single exon probes on nucleic acid microarrays and then performing two-

color fluorescent hybridization analysis; significant expression is based on a statistical confidence that the signal is significantly greater than negative biological 25 control spots. The negative biological control is formed from spotted DNA sequences from a different species. Here, 32 sequences from E.Coli were spotted in duplicate to give a total of 64 spots.

For each hybridization (each slide, each colour) 30 the median value of the signal from all of the spots is determined. The normalized signal value is the arithmetic mean of the signal from duplicate spots divided by the population median.

Control spots are eliminated if there is more 35 that a five-fold difference between each one of the

PB0004-W06-GB

duplicate spots raw signals.

The median of the signal from the remaining control spots is calculated and all subsequent calculations are done with normalised signals.

5 Control spots having a signal of greater than median + 2.4 (the value 2. 4 is roughly 12 times the observed standard deviation of control spot populations) are eliminated. Spots with such high signals are considered to be "outliers".

10 The mean and standard deviation of the modified control spot populations are calculated.

The mean + 3x the standard deviation (mean + (3*SD)) is used as the signal threshold qualifier for that particular hybridization. Thus, individual thresholds are 15 determined for each channel and each hybridization.

This means that, assuming that the data is distributed normally, there is a 99% confidence that any signal exceeding the threshold is significant.

The probes and their expression data are 20 presented in Table 4, set forth respectively in Example 5.

Example 5 presents the subset of probes that is significantly expressed in the human bone marrow and thus presents the subset of probes that was recognized to be useful for measuring expression of their cognate genes in 25 human bone marrow tissue.

The sequence of each of the exon probes identified by SEQ ID NOS.: 13,115 - 26,012 was individually used as a BLAST (or, for SWISSPROT, BLASTX) query to identify the most similar sequence in each of dbEST, 30 SwissProt (BLASTX), and NR divisions of GenBank. Because the query sequences are themselves derived from genomic sequence in GenBank, only nongenomic hits from NR were scored. The smallest in value of the BLAST (or BLASTX) 35 expect ("E") scores for each query sequence across the

PB0004-W06-GB

three database divisions was used as a measure of the "expression novelty" of the probe's ORF. Table 4 is sorted in descending order based on this measure, reported as "Most Similar (top) Hit BLAST E Value". Those sequences for 5 which no "Hit E Value" is listed are those exons which were found to have no similar sequences.

As sorted, Table 4 thus lists its respective probes (by "AMPLICON SEQ ID NO.:" and additionally by the SEQ ID NO:. of the exon contained within the probe:"EXON 10 SEQ ID NO.:") from least similar to sequences known to be expressed (i. e., highest BLAST E value), at the beginning of the table, to most similar to sequences known to be expressed (i. e., lowest BLAST E value), at the bottom of the table.

15 Table 4 further provides, for each listed probe, the accession number of the database sequence that yielded the "Most Similar (top) Hit BLAST E Value", along with the name of the database in which the database sequence is found ("Top Hit Database Source").

20 Table 4 further provides SEQ ID NOS.

corresponding to the predicted amino acid sequences where they have been determined for the probe and exon nucleotide sequences. These are set out as PEPTIDE SEQ ID NOS.:. The peptide sequences for a given exon are predicted as 25 follows: Since each chip exon is a consensus sequence drawn from predictions from various exon finding programs (i.e. Grail, GeneFinder and GenScan), the multiple initial ORFs are first determined in a uniform way according to each prediction. In particular, the reading frame for predicting 30 the first amino acid in the peptide sequence always starts with the first base of any codon and ends with the last base of non-termination codon. Next, for each strand of the exon, initial ORFs are merged into one or more final ORFs in an exhaustive process based on the following criteria: 35 1) the merging ORFs must be overlapping, and 2) the merging

PB0004-W06-GB

ORFs must be in the same frame.

The Sequence Listing, which is a superset of all of the data presented in Table 4, further includes, for each probe, the most similar hit, with accession number and 5 BLAST E value, from the each of the three queried databases. Table 4 further lists, for each probe, a portion of the descriptor for the top hit ("Top Hit Descriptor") as provided in the sequence database. For those ORFs that are 10 similar in sequence, but nonidentical to known sequences (e.g., those with BLAST E values between about le-05 and le-100), the descriptor reveals the likely function of the protein encoded by the probe's ORF.

Using BLAST E value cutoffs of le-05 (i. e., 1 x 15 10-5) and le-100 (i.e. , 1 x 10-1 ) as evidence of similarity to sequences known to be expressed is of course arbitrary: in Example 2, supra, a BLAST E value of le-30 was used as the boundary when only two classes were to be defined for analysis (unknown, le-30; known <le-30) (see also FIG. 8). 20 Furthermore, even when the "Most Similar (Top) Hit BLAST E Value" is

low, e. g., less than about le-100 - which is probative evidence that the query sequence has previously been shown to be expressed - the top hit is highly unlikely exactly to match the probe sequence.

Is First, such expression entries typically will not have the intronic and/or intergenic sequence present within the single exon probes listed in the Table. Second, even the ORF itself is unlikely in such cases to be present identically in the databases, since most of the EST and 30 mRNA clones in existing databases include multiple exons, without any indication of the location of exon boundaries.

As noted, the data presented in Table 4 represent a proper subset of the data present within the attached sequence listing. For each amplicon probe (SEQ ID NOs.: 1 35 - 13,114) and probe exon (SEQ ID NOs.: 13,115 - 26,012,

PB0004-W06-GB

: respectively), the sequence listing further provides, through iterated annotation fields <220> and <223>:

(a) the accession number of the BAC from which the sequence was derived ("MAP TO"), thus providing a link 5 to the chromosomal map location and other information about the genomic milieu of the probe sequence; (b) the most similar sequence provided by BLAST query of the EST database, with accession number and BLAST E value for the "hit"; 10 (c) the most similar sequence provided by BLAST query of the GenBank NR database, with accession number and BLAST E value for the "hit"; and À (d) the most similar sequence provided by BLASTX query of the SWISSPROT database, with accession number and 15 BLAST E value for the "hit".

EXAMPLE 5

Genome-Derived Single Exon Probes Useful For Measuring 20 Expression of Genes in Human Bone marrow Appendix 1 - Table 4 (546 pages) presents expression, homology, and functional information for the genome-derived single exon probes that are expressed significantly in 25 human bone marrow.

Claims

PB0004-WOGBI

1. A spatially addressable set of single exon nucleic acid probes for measuring gene expression in a sample derived from human bone marrow comprising a plurality of single exon nucleic acid probes, said probes comprising any one of the nucleotide sequence as set out in any of SEQ ID Nos. 450, 890, 1046, 1305, 1618, 1642, 1738, 1764, 1770, 1908,

1995, 2175, 2287, 3200, 3464, 3527, 3574, 3968, 4225, 4290,

4310, 4364, 4420, 4874, 4959, 5083, 5095, 5329, 5496, 5678,

5762, 5824, 5830, 6138, 6167, 6556, 6700, 7332, 7642, 8114,

8396, 8830, 9212, 9892, 10124, 10241, 10383, 10675, 10794,

11043, 11366, 11687, 11829, 12600, 12888, 13523, 13945,

14092, 14341, 14650, 14674, 14768, 14793, 14799, 14932,

15016, 15191, 15300, 16255, 16510, 16573, 16619, 17008,

17254, 17319, 17339, 17391, 17447, 17891, 17974, 18093,

18105, 18435, 18596, 18773, 18596, 18914, 18920, 25655,

19242, 19616, 19757, 20303, 20602, 21051, 21365, 21797,

22178, 22845, 23050, 23166, 23305, 23597, 23715, 24007,

24314, 24653, 24712, 25302 and 25476 or a complementary sequence, or a portion of such a sequence.

2. A single exon nucleic acid probe for measuring human gene expression in a sample derived from human bone marrow comprising a nucleotide sequence as set out in any of SEQ ID NOs.: 450, 890, 1046, 1305, 1618, 1642, 1738, 1764, 1770, 1908, 1995, 2175, 2287, 3200, 3464, 3527, 3574, 3968, 4225,

4290, 4310, 4364, 4420, 4874, 4959, 5083, 5095, 5329, 5496,

5678, 5762, 5824, 5830, 6138, 6167, 6556, 6700, 7332, 7642,

8114, 8396, 8830, 9212, 9892, 10124, 10241, 10383, 10675,

10794, 11043, 11366, 11687, 11829, 12600, 12888 or a complementary sequence or a fragment thereof wherein said

- -J PB0004-WO6-GBI

probe hybridizes at high stringency to a nucleic acid molecule expressed in the human bone marrow.

3. A single exon nucleic acid probe as claimed in claim 2 comprising a nucleotide sequence as set out in any of SEQ ID NOs.: 13523, 13945, 14092, 14341, 14650, 14674, 14768, 14793, 14799, 14932, 15016, 15191, 15300, 16255, 16510,

16573, 16619, 17008, 17254, 17319, 17339, 17391, 17447,

17891, 17974, 18093, 18105, 18435, 18596, 18773, 18596,

18914, 18920, 25655, 19242, 19616, 19757, 20303, 20602,

21051, 21365, 21797, 22178, 22845, 23050, 23166, 23305,

23597, 23715, 24007, 24314, 24653, 24712, 25302 and 25476 or a complementary sequence or a fragment thereof.

4. A single exon nucleic acid probe for measuring human gene expression in a sample derived from human bone marrow which is a nucleic acid molecule having a sequence encoding a peptide comprising a peptide sequence as set out in any of SEQ ID NOs.: 26455, 26903, 27305, 27626, 27647, 27753, 27778, 27785, 27928, 28023, 28212, 28324, 29175, 29431,

29496, 30141, 30198, 30218, 30219, 30338, 30780, 30865,

30969, 30980, 31187, 31188, 32097, 32103, 32443, 32473,

33034, 33035, 33647, 33648, 33966, 33967, 34774, 35218,

35608, 35609, 36302, 36529, 36653, 36782, 37093, 37094,

37216, 37217, 38232 and 31730 or a complementary sequence or a fragment thereof wherein said probe hybridizes at high stringency to a nucleic acid expressed in the human bone marrow. 5. A single exon nucleic acid probe as claimed in any one of claims 2 to 4 wherein said single exon nucleic acid probe comprises between 15 and 25 contiguous nucleotides of

PB0004-WO6-GBI

said SEQ ID NO.

6. A single exon nucleic acid probe as claimed in any one of claims 2 to 4, wherein said probe is between 3 - 25 kb in length.

7. A single exon nucleic acid probe as claimed in any one of claims 2 - 6, wherein said probe is DNA, RNA or PEA.

8. A single exon nucleic acid probe as claimed in any one of claims 2 - 7, wherein said probe is detectably labeled.

9. A single exon nucleic acid probe as claimed in any one of claims 2 - 8, wherein said probe lacks prokaryotic and bacteriophage vector sequence.

10. A single exon nucleic acid probe as claimed in any one of claims 2 9, wherein said probe lacks homopolymeric stretches of A or T. 11. A nucleic acid sequence as set out in any of SEQ ID NOs: 450, 890, 1046, 1305, 1618, 1642, 1738, 1764, 1770, 1908,

1995, 2175, 2287, 3200, 3464, 3527, 3574, 3968, 4225, 4290,

4310, 4364, 4420, 4874, 4959, 5083, 5095, 5329, 5496, 5678,

5762, 5824, 5830, 6138, 6167, 6556, 6700, 7332, 7642, 8114,

8396, 8830, 9212, 9892, 10124, 10241, 10383, 10675, 10794,

11043, 11366, 11687, 11829, 12600, 12888, 13523, 13945,

14092, 14341, 14650, 14674, 14768, 14793, 14799, 14932,

15016, 15191, 15300, 16255, 16510, 16573, 16619, 17008,

17254, 17319, 17339, 17391, 17447, 17891, 17974, 18093,

18105, 18435, 18596, 18773, 18596, 18914, 18920, 25655,

19242, 19616, 19757, 20303, 20602, 21051, 21365, 21797,

PB0004-WO6-GBI

22178, 22845, 23050, 23166, 23305, 23597, 23715, 24007,

24314, 24653, 24712, 25302 and 25476 which encodes a peptide.

12. A peptide encoded by a sequence as set out in any of SEQ ID Nos: 450, 890, 1046, 1305, 1618, 1642, 1738, 1764, 1770, 1908, 1995, 2175, 2287, 3200, 3464, 3527, 3574, 3968,

4225, 4290, 4310, 4364, 4420, 4874, 4959, 5083, 5095, 5329,

5496, 5678, 5762, 5824, 5830, 6138, 6167, 6556, 6700, 7332,

7642, 8114, 8396, 8830, 9212, 9892, 10124, 10241, 10383,

10675, 10794, 11043, 11366, 11687, 11829, 12600, 12888,

13523, 13945, 14092, 14341, 14650, 14674, 14768, 14793,

14799, 14932, 15016, 15191, 15300, 16255, 16510, 16573,

16619, 17008, 17254, 17319, 17339, 17391, 17447, 17891,

17974, 18093, 18105, 18435, 18596, 18773, 18596, 18914,

18920, 25655, 19242, 19616, 19757, 20303, 20602, 21051,

21365, 21797, 22178, 22845, 23050, 23166, 23305, 23597,

23715, 24007, 24314, 24653, 24712, 25302 and 25476.

13. A peptide comprising a sequence as set out in any of SEQ ID Nos: 26455, 26903, 27305, 27626, 27647, 27753, 27778, 27785, 27928, 28023, 28212, 28324, 29175, 29431, 29496,

30141, 30198, 30218, 30219, 30338, 30780, 30865, 30969,

30980, 31187, 31188, 32097, 32103, 32443, 32473, 33034,

33035, 33647, 33648, 33966, 33967, 34774, 35218, 35608,

35609, 36302, 36529, 36653, 36782, 37093, 37094, 37216,

37217, 38232 and 31730.

14. A microarray comprising a spatially-addressable set of single exon nucleic acid probes for measuring gene expression in a sample derived from human bone marrow comprising a plurality single exon nucleic probes, said probes comprising any one of the nucleotide sequences set

PB0004-WO6-GBI

out in SEQ ID NOs: 1 - 13,114 or a complementary sequence, or a portion of such a sequence.

15. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in claim 14 wherein each of said plurality of probes is separately and addressably amplifiable.

16. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in claim 14 or 15 wherein each of said plurality of probes is separately and addressably isolatable from said plurality.

17. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in any of claims 14 to 16 wherein said probes comprise any one of the nucleotide sequences set out in SEQ ID NOS. : 13,115 -

26,012.

18. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in any of claims 14 to 17, wherein each of said plurality of probes is amplifiable using at least one common primer.

19. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in any of claims 14 to 18 wherein the set comprises between 50 - 20,000 single exon nucleic acid probes.

20. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in any of claims 14 to 19, wherein the average length of the single exon

- J PB0004-WO6-GBI

nucleic acid probes is between 200 and 500 bp.

21. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in any of claims 14 to 20, wherein at least 50% of said single exon nucleic acid probes lack prokaryotic and bacteriophage vector sequence. 22. A microarray comprising a spatiallyaddressable set of single exon nucleic acid probes as claimed in any of claims 14 to 21, wherein at least 50% of said single exon nucleic acid probes lack homopolymeric stretches of A or T. 23. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in any of claims 14 - 22 characterized in that said set of probes is addressably disposed upon a substrate.

24. A microarray comprising a spatially-addressable set of single exon nucleic acid probes as claimed in claim 23 wherein said substrate is selected from glass, amorphous silicon, crystalline silicon and plastic.

25. A spatially addressable set of single exon nucleic acid probes for measuring gene expression in a sample derived from human bone marrow comprising a plurality of single exon nucleic acid probes, said probes comprising any one of the nucleotide sequences set out in SEQ ID Nos. 1 26,012 or a complementary sequence, or a portion of such a sequence. 26. A single exon nucleic acid probe for measuring human

PB0004-WO6-GB1

gene expression in a sample derived from human bone marrow comprising a nucleotide sequence as set out in any of SEQ ID NOs.: 1 - 13,114 or a complementary sequence or a fragment thereof wherein said probe hybridizes at high stringency to a nucleic acid molecule expressed in the human bone marrow.

27. A single exon nucleic acid probe as claimed in claim 26 comprising a nucleotide sequence as set out in any of SEQ ID NOs.: 13,115 - 26,012 or a complementary sequence or a fragment thereof.

28. A single exon nucleic acid probe for measuring human gene expression in a sample derived from human bone marrow which is a nucleic acid molecule having a sequence encoding a peptide comprising a peptide sequence as set out in any of SEQ ID NOs.: 26,013 - 38,628, or a complementary sequence or a fragment thereof wherein said probe hybridizes at high stringency to a nucleic acid expressed in the human bone marrow.

29. A single exon nucleic acid probe as claimed in any one of claims 26 to 28 wherein said single exon nucleic acid probe comprises between 15 and 25 contiguous nucleotides of said SEQ ID NO.

30. A single exon nucleic acid probe as claimed in any one of claims 26 to 28, wherein said probe is between 3 - 25 kb in length.

31. A single exon nucleic acid probe as claimed in any one of claims 26 30, wherein said probe is DNA, RNA or PNA.

- PB0004-WC6-GBI

32. A single exon nucleic acid probe as claimed in any one of claims 26 31, wherein said probe is detectably labeled.

33. A single exon nucleic acid probe as claimed in any one of claims 26 32, wherein said probe lacks prokaryotic and bacteriophage vector sequence.

34. A single exon nucleic acid probe as claimed in any one of claims 26 33, wherein said probe lacks homopolymeric stretches of A or T. 35. A method of measuring gene expression in a sample derived from human bone marrow, comprising: contacting the microarray of any of claims 14 -24, with a first collection of detectably labeled nucleic acids, said first collection of nucleic acids derived from mRNA of human bone marrow; and then measuring the label detectably bound to each probe of said microarray.

36. A method of identifying exons in a eukaryotic genome, comprising: algorithmically predicting at least one exon from genomic sequence of said eukaryote; and then detecting specific hybridization of detectably labeled nucleic acids to a single exon probe, wherein said detectably labeled nucleic acids are derived from mRNA from the bone marrow of said eukaryote, said probe is a single exon probe having a fragment identical in sequence to, or complementary in sequence to, said predicted exon, said probe is included within a microarray

PB0004-WO6-GBI

according to any of claims 14 - 24, and said fragment is selectively hybridizable at high stringency.

37. A method of assigning exons to a single gene, comprising: identifying a plurality of exons from genomic sequence according to the method of claim 36; and then measuring the expression of each of said exons in a plurality of tissues and/or cell types using hybridization to single exon microarrays having a probe with said exon, wherein a common pattern of expression of said exons in said plurality of tissues and/or cell types indicates that the exons should be assigned to a single gene.

38. A nucleic acid sequence as set out in any of SEQ ID NOs: 1 - 26,012 which encodes a peptide.

39. A peptide encoded by a sequence as set out in any of SEQ ID Nos: 1 26,012.

40. A peptide comprising a sequence as set out in any of SEQ ID Nos: 26, 013 - 38,628.