WO2002018655A2

WO2002018655A2 - Compositions and methods for the generation of single stranded cdna microarrays with accurate universal quantitation reference

Info

Publication number: WO2002018655A2
Application number: PCT/US2001/027021
Authority: WO
Inventors: Wei Zhang; Limei Hu; Stanley R. Hamilton
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2000-09-01
Filing date: 2001-08-30
Publication date: 2002-03-07
Also published as: WO2002018655A3; AU2001286929A1

Abstract

Single stranded nucleic acid oligonucleotides are immobilized on a solid support to provide a DNA chip, or microarray of single stranded DNA immobilized on a solid support, for hybridizing oligonucleotides. A universal and quantitatively reproducible hybridization reference oligonucleotide is used for data normalization and for quality control of the array printing process.

Description

COMPOSITIONS AND METHODS FOR THE GENERATION OF SINGLE

STRANDED cDNA MICROARRAYS WITH ACCURATE UNIVERSAL

QUANTITATION REFERENCE

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods for DNA chips or DNA microarrays immobilized on a solid support for hybridizing oligonucleotides and in particular to single stranded cDNA microarrays together with a universal and quantitatively reproducible hybridization reference. BACKGROUND OF THE INVENTION Without limiting the scope of the invention, its background is described in connection with compositions and methods for DNA chips or DNA microarrays immobilized on a solid support, as an example.

The expression levels of various genes ar^*e indicative of many disease states. Differences in the copy number of the genetic DNA or through changes in levels of transcription (e.g. through control of initiation, provision of RNA precursors, RNA processing, etc.) of particular genes are often characteristic of disease. Losses and gains of genetic material, for example, play an important role in malignant transformation and progression. Changes in the expression of oncogenes or tumor suppressor genes may be diagnostic or predictive of such malignant transformation. Oncogenes are positive regulators of tumorgenesis . Tumor suppressor genes are negative regulators of tumorgenesis (Marshall, Cell, 64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991)). An increase the number of genes coding for oncogene proteins or an increase in the level of expression of these oncogenes (e.g. in response to changes of the cell or environment) , often result in unregulated cellular growth. Similarly, the loss of genetic material or a decrease in the level of expression of genes that code for tumor suppressors may lead to malignancy. For example, it has been shown that losses and gains of genetic material are associated with glioma progression (Mikkelson et al . J. Cellular Biochm. 46: 3-8 (1991)). Changes in the expression (transcription) levels of particular genes (e.g. oncogenes or tumor suppressors), are therefore well recognized indicators of the presence and growth of various cancers. The cell cycle and cell development are also characterized by the variations in the transcription levels of particular genes. A viral infection, for example, is often characterized by elevated expression of genes of the particular virus. Outbreaks of many disease-causing viruses, such as Herpes simplex, Epstein-Barr virus infections (e.g. infectious mononucleosis) , cytomegalovirus, Varicella-zoster virus infections, parvovirus infections, human papillomavirus infections and others, are characterized by elevated expression of genes present in the respective virus. An effective diagnostic of the disease state caused by the virus is to detect elevated expression levels of characteristic viral genes. Further, many viruses such as herpes simplex, enter quiescent states for periods of time only to break out in explosive periods of rapid replication. The detection of expression levels of characteristic viral genes allows diagnosis of such proliferative and infective activity of the virus. Oligonucleotide probes have long been used to detect complementary nucleic acid sequences in a nucleic acid of interest (the "target", "test" or "experimental" nucleic acid) and have been used to detect expression of particular genes (e.g., a Northern Blot). In some assay formats, the oligonucleotide probe is immobilized or tethered, i.e., by covalent attachment, to a solid support, and arrays of oligonucleotide probes so immobilized on solid supports have been used to detect specific nucleic acid sequences in a target nucleic acid. See, e.g., PCT patent publication Nos. WO 89/10977 and 89/11548. The use of oligonucleotide probes has been proposed to sequence a target nucleic acid. See U.S. Pat. Nos. 5,202,231 and 5,002,867 and PCT patent publication No. WO 93/17126.

The development of VLSIPS^® technology provided methods for synthesizing arrays of many different oligonucleotide probes that occupy a very small surface area and that can be used to detect the presence of a nucleic acid containing a specific nucleotide sequence. See U.S. Pat. No. 5,143,854 and PCT patent publication No. WO 90/15070.

Prior methods for constructing a cDNA microarray involve depositing or printing double stranded cDNA products onto glass slides coated with a substance such as poly-lysine, followed by ultraviolet (UV) cross- linking. Printing refers to any of a variety of means known to those of skill for immobilizing nucleic acid on a solid support in a pattern or array. U. S. Pat. No. 5,919,626, issued July 6, 1997 to Shi, et al., for example, describes a variety of methods to immobilize nucleic acid on a solid support, including ink-jet printing. During the hybridization procedure, the slides with immobilized double stranded (ds) DNA oligonucleotides are heated briefly to denature and separate the double strands into single strand molecules. For example, Shalon, et al., place the slides in distilled water at 90°C for two minutes to denature the double stranded DNA immobilized on the slide. Genome Res. 6(7): 639-45, July, 1996. See also U. S. Pat. No. 5,985,567, issued November 15, 1997 to Ra pal . Hybridization occurs upon exposure of the immobilized DNA, under conditions known to those of skill, with the test sample cDNA that is labeled with a fluorescent dye such as Cy₃ or Cy₅. The efficiency is so low, however, that the procedure requires 50-100 micrograms of total RNA to get a sufficient signal.

Another problem with prior art DNA chips or microarrays is the lack of a universal reference for quantitation of the array results. A reference RNA sample of the prior art is typically from a mixture of a series of established cell lines. See, for example, U.S. Pat. No. 6,013,449 issued January 11, 2000 to Hacia, et al., where reference nucleic acid sequences are described as being derived from human genes associated with genetic disease and include genes for BRCA-1, BRCA-2, p53, N-,C- and K-ras, cytochromes p450, CFTR, HLA classes I and II, and β-Globin.

The reference RNA is used to generate reference cDNAs . The reference RNA is labeled with a first dye such as Cy3, and the test samples are labeled with a second dye such as Cy5. The test sample is typically cDNA derived from mRNA expressed by a gene of interest. The reference sample and the test sample are cohybridized to the same microarray slide. For each single gene, which is represented by each spot on the array, the ratio of the intensities from the two dyes provides the quantitation of the expression level of the gene of interest.

There are several problems associated with the prior art approach. First, it is difficult to standardize among different investigators because each one has to use the same reference sample in order to compare results. Variations in the mRNA population within the reference sample is virtually unavoidable, even where different investigators use the same sample, because the effects of time, transport, laboratory handling and other factors produce such variations. Second, even with a mixture of cell lines, many genes are not expressed in the reference sample, rendering the ratio-taking impossible because the test sample cannot find a complementary strand on the array with which to hybridize and produce a signal.

SUMMARY OF THE INVENTION

The present invention overcomes the problems just described with the prior art. To overcome the inefficient heating step of the prior art to denature the immobilized double stranded DNA, the present invention deposits single stranded cDNA, instead of double stranded DNA, on the glass matrix. The single stranded cDNA is obtained by either asymmetric polymerase chain reaction (PCR) or a biotin-strepavidin based strategy.

The present invention involves isolating single stranded cDNA and then depositing the cDNA strands on coated glass slides to form the cDNA microarrays. There are two ways to isolate single stranded DNA: 1) asymmetric PCR, where one strand of DNA can be enriched by using only one PCR primer, or, 2) performing regular PCR with one primer end-labeled with biotin. The product is then heated to 90°C, and the biotin-labeled strand is removed by strepavidin beads or strepavidin coated tubes and the remaining single strand is transferred to a new tube .

To overcome prior art problems with the reference samples, the present invention provides compositions and methods for a labeled universal reference oligonucleotide that can hybridize with every target gene on the array, providing a reproducible reference point for ratio- taking. That is, every single stranded probe immobilized on the array contains a predetermined oligonucleotide sequence complementary to the reference oligonucleotide, and the complementary sequence is the same on each probe. The reference serves as a normalizer that controls for uneven ^"hybridization and washing that lead to uneven background. Further, a reference oligonucleotide of the present invention also can be used to monitor and control the quality of the DNA printing process.

The probe oligonucleotides containing a predetermined sequence complementary to the reference sequence of the present invention may be chemically synthesized from the solid support by chemically adding oligonucleotides in a desired sequence from an initial oligonucleotide anchored to the support. Such chemical synthesis of the probe with a predetermined sequence complementary to the reference sequence may have advantages over PCR amplification to produce such probes, and may be a preferred embodiment of the present invention for some investigators .

The present invention provides a kit for oligonucleotide hybridization, including an array of nucleic acids immobilized on a solid support where each of the immobilized nucleic acids includes a predetermined sequence. A first labeled oligonucleotide complementary to the predetermined sequence of the immobilized nucleic acids is also included. The labeled oligonucleotide hybridizes with every nucleic acid on the array.

Another embodiment of the kit of the present invention includes an array of single stranded oligonucleotide probes immobilized on a solid support. The immobilized oligonucleotide probes are the product of amplification by polymerase chain reaction using a predetermined primer oligonucleotide. Also included in the kit is a detectable reference oligonucleotide complementary to the primer oligonucleotide. The reference oligonucleotide hybridizes with every probe on the array and is distinguishable from a test oligonucleotide hybridized to the array. In yet another embodiment of the kit, the kit includes one or more reagents to produce one or more nucleic acids where at least one of the reagents includes an oligonucleotide having a predetermined sequence that is incorporated into each of the nucleic acids. A solid support is provided for immobilizing the single stranded nucleic acids to provide an array of single stranded nucleic acids immobilized on the solid support. The kit has a detectable oligonucleotide complementary to the predetermined sequence and that hybridizes with every nucleic acid on the array.

The present invention also contemplates a system for oligonucleotide hybridization. The system includes an array of single stranded nucleic acids immobilized on a solid support, where the immobilized nucleic acids have a predetermined sequence. A labeled oligonucleotide is provided that hybridizes to the predetermined sequence present in each nucleic acid of the array to provide a quantitatively reproducible baseline signal for normalizing signals obtained from hybridization with the array.

Another embodiment of the system for oligonucleotide hybridization includes one or more reagents to produce single stranded nucleic acids. The reagents include a primer oligonucleotide with a predetermined sequence for incorporation into each of the single stranded nucleic acids. A solid support for immobilizing the single stranded nucleic acids is included in the system to provide an array of single stranded nucleic acids immobilized on the solid support. A detectable oligonucleotide complementary to the predetermined sequence and that hybridizes with every nucleic acod on the array and wherein the detectable oligonucleotide is also included in the system.

The present invention provides methods for hybridizing oligonucleotides. One method of the invention includes immobilizing one or more nucleic acids on a solid support. Each of the one or more the single stranded nucleic acids includes a predetermined sequence. The method further includes the step of providing a first labeled oligonucleotide complementary to the predetermined sequence so that the first labeled oligonucleotide hybridizes with every nucleic acid on the solid support at the predetermined sequence to provide a quantitatively reproducible baseline signal for normalizing signals obtained from hybridization with the nucleic acids on the solid support.

In another embodiment, the method includes the further steps of providing a second labeled oligonucleotide to provide a second signal; exposing the first labeled oligonucleotide and the second labeled oligonucleotide to the nucleic acids on the solid support under conditions permissive for hybridization; removing nonspecifically bound substances from the solid support; detecting the signal from the first label and the second label from the first and second labeled oligonucleotides hybridized to the nucleic acids on the solid support to obtain a ratio; and normalizing the ratio to the quantified signal of the first label.

In yet another embodiment of the method of the present invention for hybridizing oligonucleotides, instead of an array of immobilized probes, single stranded oligonucleotide probes and a solid support for immobilizing the single stranded oligonucleotide probes are provided so that one may immobilize the single stranded oligonucleotide probes on the support to produce the array of immobilized probes. Otherwise, the method is as described above.

The present invention provides a more efficient cDNA microarray system to obtain more accurate measurements of gene expression levels than is provided by the prior art, rendering current commercial microarray technologies obsolete .

BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures in which corresponding numerals in the different figures refer to corresponding parts and in which:

Figure 1 is a fluorograph showing superimposed fluorescently labeled DNA arrays of the present invention.

Figure 2a is an autofluorograph of a gel showing the yield of single stranded DNA by different methods of the present invention.

Figure 2b is a fluorograph of an array showing Cys labeled reference primer of the present invention.

Figure 2c is a fluorograph of an array showing Cy₃ labeled reference primer of the present invention.

Figure 2d is a composite fluorograph image of Figs. 2b and 2c.

Figure 3 is fluorographs of three different arrays hybridized with fluorescent Cy₅-labeled reference primer of the present invention. DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts which can be embodied in a wide variety of specific contexts . The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. DNA clones referred to herein are commercially available unless otherwise indicated.

DNA inserts for 30,000 clones obtained from Research Genetics, Inc., Huntsville, AL, were amplified by the same set of primers to produce cDNA oligonucleotides strands for use as probes to be printed on the array. The orientation of the cDNA strands, however, is random. There are several ways to address the orientation of the cDNAs . One way is to predetermine the orientation of all the clones by methods known to those skilled in the art and to use correct primers to amplify the sense strand before printing.

Alternatively, both strands of the cDNA in the test samples may be amplified in a linear fashion following the SMART^® procedure of Clontech Laboratories, Inc., Palo Alto, CA, with both primers labeled with the same fluorescent dye. Third, both strands may be isolated and a pair of twin single strand DNA microarrays can be made. An advantage of the third option is that for each clone, only one of the arrays should give a positive signal following the conventional procedure known to those skilled in the art where mRNA is reversed to cDNA for hybridization. The pattern obtained thereby serves as a control for the specificity of hybridization.

Another method of eliminating one of the two natural stands of the target DNA molecule from the reaction to produce single-stranded DNA molecules may be produced using the single-stranded DNA bacteriophage M13 (Messing, J. et al., Meth. Enzymol . 101:20 (1983); see also, Sambrook, J. , et al . (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) ) .

Quality control of the printing process and quantitation of the array hybridization results are easily accomplished with the present invention. The primer used for amplifying the single strand DNA to be printed on the glass is labeled with FITC for quality control of the printing process. After printing of the slides, the slides can be scanned by a laser imager to monitor the evenness of the printing process.

A complementary oligonucleotide corresponding to the primer is made and labeled with a first stable fluorescent dye such as Cy₃ for hybridization. The Cy₃- labeled oligonucleotide serves as a universal reference sequence because it can hybridize with all the clones on the array equally. The reference oligonucleotide may be made, for example, by methods of chemical oligonucleotide synthesis known to those of skill in the art. See U.S. Pat. No. 5,837,832, issued November 17, 1995 to Chee, et al . Alternatively, or additionally, reference oligonucleotides of the present invention may be made by biological methods of oligonucleotide synthesis known to those of skill in the art.

The test sample is produced by reverse transcription of the mRNA of a gene of interest and amplified by the commercially available SMART^® procedure (Clontech Laboratories, Inc.). The experimental sample is labeled with a second fluorescent dye, different from that of the reference sample, such as Cy₅. Briefly, the SMART procedure is described as one method of obtaining labeled DNA. SMART^® cDNA synthesis begins with just nanograms of either total or poly A+ RNA, with a modified oligo(dT) primer is used to prime the first-strand reaction. When reverse transcriptase (RT) reaches the 5' end of the mRNA, the enzyme's terminal transferase activity adds a few deoxycytidine (dC) nucleotides. The 3' end of the SMART^® oligonucleotide anneals with the (dC) stretch, forming an extended template. Reverse transcriptase then switches templates and replicates the oligonucleotide. The resulting single-stranded (ss) cDNA contains the complete 5' end of the mRNA template, as well as the sequence complementary to the SMART oligonucleotide, e.g., the SMART anchor. The anchor, together with the modified oligo (dT) sequence, serves as a universal priming site for long-range PCR, primer extension, or RACE amplification.

Fluorochromes or dyes for use with the present invention will depend on wavelength and coupling structure compatibility. By means of example, FITC, Fluorescein-5-EX, 5-SFX, Rhodamine Green-X, Bodipy FL-X, Cy2-OSu, Fluor X, 5(6)TAMRA-X, Bodipy TMR-X, Rhodamine Red-X, Texas Red-X, Bodipy TR-X, Cy3-OSu, Cy3.5-OSu, Cy5- Osu and/or Cy5.5-OSu, may be used if desired. In one method of the present invention, the reference sample is aliquoted and one aliquot is used per study. The reference oligonucleotide sample is mixed with the test sample. The mixture is then cohybridized to the array.

The reference oligonucleotide hybridizes with all the clones on the array. Variations between assays in the fluorescent signals after hybridization and wash are normalized according to the reference sample fluorescent signal. A ratio is taken between the normalized Cy₅ and Cy₃ signals for each gene of each test sample.

In contrast to radioactive labels and chemiluminescent labels which may be used in the present invention, an advantage of fluorescent labels is that signals from fluorescent labels do not disperse. The lack of dispersal in the fluorescent signal permits dense spacing of probes on the array. Another advantage of fluorescent probes is that the signal from a reference sample or a test sample hybridized to a probe on the array can be detected separately. Multiple-color hybridization detection permits direct quantitative determination of the relative abundance of sequences hybridized to the array.

For arrays fabricated on glass slides, it is known that increasing the hydrophobicity of the glass surface enhances DNA printing by permitting relatively more densely packed arrays. Poly- -lysine coated slides are thus commonly used for printing nucleic acid arrays. Poly-lysine coated slides may be fabricated in the laboratory by methods known to those of skill in the art, but they are also commercially available from, for example, Sigma Chemical Co., Inc. Several methods may be used to immobilize one or more of the nucleic acid reactants to a solid support. For example, a 96-well polystyrene plates are widely used in solid-phase immunoassays, and several PCR product detection methods that use plates as a solid support. The most specific of these methods require the immobilization of a suitable oligonucleotide probe into the microtiter wells followed by the capture of the PCR product by hybridization and colorimetric detection using, e.g., biotin-avidin.

The means by which macromolecules bind non- covalently to, e.g., polystyrene and glass surfaces is not well understood. Nevertheless, the observation that nucleic acids to bind to these surfaces, and that the binding may be enhanced using, e.g., poly-L-Lysine, has proven to be important in the development and manufacturing of nucleic acid detection arrays and assay, including diagnostic tests where one component needs to be immobilized and normalized across samples and between tests. In addition to poly-L-lysine, glass slides may be treated by silanation or silynation.

Polystyrene is a hydrophobic material suitable for binding negatively charged macromolecules because it normally contains few hydrophilic groups. Microtiter plate manufacturers have developed methods of introducing such groups (hydroxyl, carboxylate and others) onto the surface of microwells to increase the hydrophilic nature of the surface. Theoretically, this allows macromolecules to bind through a combination of hydrophobic and hydrophilic interactions (Baier et al., Science 162: 1360-1368 (1968); Baier et al., J. Biomed. Mater. Res. 18: 335-355 (1984); Good et al., in L. H. Lee (ed. ) Fundamentals of Adhesion, Plenum, New York, chapter 4 (1989)). In practice, some proteins bind more efficiently to the treated hydrophilic polystyrene than to the untreated material, due to non-polar interactions. In some embodiments of the present invention, for example when capturing a nucleic acid with a DNA binding protein, the binding of a protein to an untreated polystyrene surface may be used. Covalent binding to polystyrene, e.g., microtiter wells, has proven to be inefficient, therefore passive adsorption remains the most commonly used method of binding macromolecules to such wells. The term "polystyrene" may also be used to describe styrene-containing copolymers such as: styrene/divinyl benzene, styrene/butadiene, styrene/vinyl benzyl chloride and others.

While polystyrene is an organic hydrophobic substrate, glass provides an inorganic hydrophilic surface. The most common glass format in assays is the microscope slide. Laboratory-grade glasses are predominantly composed of Si0₂, however, the glass may be doped with charged molecules, e.g., metal-based silica oxides . Interfaces involving such materials provide modified surfaces that may be used to incorporate the bulk properties of different phases into a uniform composite structure. In addition to glass and plastic (polystyrene) substrates, metal substrates including metal slides may also be suitable for the present invention.

Double stranded cDNA molecules have been used to generate cDNA microarrays. To hybridize a strand of probe DNA on the array to the test DNA pursuant to the prior art, a heating step is commonly used to separate the strands of the double stranded DNA on the array. The efficiency of this method, however, is very low. The present invention allows a complete circumvention of this inefficient step.

With the present invention, all the DNA molecules on the array are available for hybridization. The increased efficiency of the present invention allows a very small amount of total RNA to be used for generating a high signal-noise ratio from a single strand array.

Prior art methods for quantifying gene expression on a glass-based cDNA array rely on taking a ratio against a reference RNA sample that is derived from a series of up to ten different cell lines. The prior art approach is very hard to standardize. For example, the same ten cell lines from different labs can vary greatly. It is difficult to produce enough RNA for multiple projects that can last for long period of time.

The present invention overcomes the problem. The reference oligonucleotide of the present invention is a universal one and is chemically or biologically synthesized by methods known to those skilled in the art. Thus, the quality and quantity of the reference signal are easily standardized. The universal reference assures a consistent and quantitatively reproducible baseline signal for all the genes on the array, enabling one to take a ratio for all the genes on the array.

The primer oligonucleotide from which the reference oligonucleotide is derived may be any one of a variety of polymerase chain reaction initiation sequences known to those skilled in the art. The sequences from which the reference oligonucleotide may be derived include the T3 transcription initiation sequence, the T7 transcription initiation sequence, the Sp6 transcription initiation sequence, the M13 transcription initiation sequence and the consensus translation initiation sequence GCCA/GCCATGG.

The probe oligonucleotides containing a predetermined sequence complementary to the reference sequence of the present invention may be chemically synthesized from the solid support by chemically adding oligonucleotides in a desired sequence from an initial oligonucleotide anchored to the support. Such chemical synthesis of the probe with a predetermined sequence complementary to the reference sequence may have advantages over PCR amplification to produce such probes, and may be a preferred embodiment of the present invention for some investigators.

The probe may be engineered such that the predetermined sequence complementary to the reference sequence of the present invention is at any desired location within the probe oligonucleotide. One the one hand, one may locate the reference sequence complementary to the predetermined sequence at the base of the probe, at the end of the probe that is anchored to the support, to protect the sequence from degradation which may occur if the sequence is located at the free end of the probe. On the other hand, one may desire to locate the predetermined sequence complementary to the reference sequence at the free end of the probe to enhance its ability to hybridize with the reference oligonucleotide by avoiding steric or other hindrances that may exist for sequences near the base of the probe. The predetermined sequence complementary to the reference sequence, of course, may also be located at any point in between these two extremes, provided, however, that the sequence predetermined complementary to the reference sequence is not located within the probe so as to prevent hybridization of the experimental sample to a complementary probe on the array.

Prior art approaches do not allow data normalization across the whole area of the array. The present invention, using a universal reference oligonucleotide, allows such normalization. The accuracy of gene expression measurement with the present invention is thereby greatly improved over the prior art.

To illustrate the invention just described, reference is now made to the Figures.

Figure 1 illustrates the specificity of hybridization with the present invention. More than 100 cDNAs were spotted on poly-lysine coated slides using GeneTAC G³ Robotic Workstation from Genomic Solutions, Inc., Ann Arbor, MI. The Genomic Solutions, Inc., spotter uses solid titanium pins, rather than a microvalve, to spot the array. The slides were hybridized with reference primer labeled with Cys, generating an image that fluoresces red, and with an test probe, IGFBP2, labeled with Cy₃, generating an image that fluoresces green. The two images are superimposed in Fig. 1. Only the cDNAs corresponding to IGFBP2 genes were detected by Cy₃ labeled IGFBP2.

Figures 2a-2d illustrate that single strand cDNAs arrayed on a solid support of the present invention produce markedly strong signals. In Fig. 2a, DNA strands amplified by either asymmetric PCR where both double stranded DNA (ds) and single stranded DNA (ss) were produced, or by regular PCR where only double strand DNA was produced, were fluorescently labeled and run on a gel. Lanes designated HI and FI refer to particular DNA clones that were amplified. The yield of asymmetric PCR roughly corresponds to band thickness and/or brightness. The yield of asymmetric PCR is generally less than with regular PCR because only one primer is used.

In Fig. 2b, the asymmetric PCR and regular PCR products from the clones Hi and FI of Fig. 2a, were spotted on poly-lysine coated glass slides together with many other clones to obtain a DNA array of the present invention. The slide was cohybridized with Cy₅-labeled reference primer and Cy₃ labeled cDNA from a tumor cell line. Fig. 2b shows the array as viewed with a Cy₅ channel imager. Substantially more DNA strands are detected with ds PCR product than ss PCR product.

Fig. 2c shows the co-hybrid array of Fig. 2b, now viewed with a Cy₃ channel imager. The Cy₃ channel shows a similar intensity from ds and ss DNAs, although there are much more ds DNAs on the slide. Fig. 2c demonstrates that the single stranded DNA is more efficient for generating a signal than is double stranded DNA.

Fig. 2d is a composite image with the Cy₃ and Cy₅ channels superimposed. Hybridization may be quantified by determining the ratio of the Cy₃ signal to the Cy_s signal .

Figure 3 shows three different arrays of the present invention hybridized with a Cys-labeled reference primer of the present invention. A very consistent hybridization pattern is seen. Variable intensities are also seen with some spots. This demonstrates that the reference primer can be used to monitor the uniformity and reproducibility of DNA spotting and hybridization.

The results illustrated in Figures 1-3 demonstrate three points. First, the microarray procedure of the present invention is highly specific. Second, single stand cDNAs arrayed on the slides produce much stronger signal than corresponding double strand cDNA printed on the same slide. Third, the universal primer of the present invention serves as an excellent reference for data normalization and for quality control of the array printing process. The present invention is capable of being used with multiple fluorescence wavelength detection systems, for example, by filtering different wavelengths or by other hyperspectral methods. One example of a use for the present invention is the expression level analysis of, e.g., 10,000 or more independent samples deposited or created on slides. To deposit that many samples of a single slide, a microchemical spotting system may be used. Such systems are presently used at Stanford University, California, or from Synteni, U.S.A. Alternatively, other slide spotting systems may be built using array technologies such a photolithographic techniques and photodeprotection chemistry.

High density arrays of oligonucleotide (or other) probes are an emerging technology for research and potential clinical diagnostics. Arrays of up to 65,000 oligos, manufactured using photolithographic methods are now available commercially from Affymetrix/Hewlett Packard. These arrays are used for resequencing and expression studies via hybridization to the array. These chips currently have feature sizes of 20 micron. The present invention provides methods and compositions that will improve the performance of sample analysis in such systems by allowing normalization of the results read from such machines, including the capacitance-coupled systems by providing a reference oligonucleotide that normalizes the signal for binding among difference chips or slides.

The present invention may be used with slide spotter systems in conjunction with a high-throughput reader analysis for gene expression determinations such as the GeneTAC G³ Robotic Workstation from Genomic Solutions, Inc., Ann Arbor, MI. For example, the present invention may be used to normalize expression data to measure the expression level of all 6,217 genes (ORFs) in yeast in response to knocking-out each gene, thus creating a 6,217 x 6,217 array of expression results, from which the gene networks will be computed. Like studies may be conducted, and relative expression levels determined for all organisms with large numbers of known genes.

A slide spotter such as the GeneTAC G³ Robotic Workstation from Genomic Solutions, Inc., Ann Arbor, MI., which uses solid pins with a "dip and print" technique using about InL of sample for each feature on a slide may be used to spot an array of the present invention. Alternatively, a slide spotter may be constructed from, e.g., a Toshiba high precision/reproducibility pick and place robot with a multi-channel spotting head. The Toshiba robot is programmable from a teach pendant or via PC computer. Different types of print heads may be used to spot slides, e.g., a pin spotter, a microvalve/capillary spotter or a piezoelectric/capillary spotter. These provide options of increasing accuracy, complexity and risk. An ultra clean environment is maintained using a HEPA filter to pressurize robot operating volume and proper clean room practices. Microwell plates are kept cool using a surface chiller to minimize evaporation.

Specifications for a slide spotter can include a spot volume of 500 picoliters to 10 nanoliters, a total volume deposited of 500 picoliters (if used with 40 slides this requires 20 nanoliters to 400 nanoliters of volume) , and a total sample prime volume of 2 microliters. A drop size for use with slide spotting may be 90 picoliters (e.g., a piezo shooter system, 0.5-1.0 nanoliters for microvalve, or 1-10 nanoliters for pin tool) . The system should provide a spot reproducibility of approximately > 95%. Shoot times of 6 milliseconds (piezo) to 0.1 seconds (microvalve or pin tool) may be used. Spot dimensions may be of up to about 100 microns on a slide size of, e.g., one inch x three inches. A post grid or orientation may be of 48 x 144 post, with a slide spot area of 0.75 x 2.25 inches (about 19 mm x 57 mm) . The distance between spots may be of about 0.19 mm/48 spots which totals 396 microns. The X-Y step size and reproducibility of a Toshiba robot is about 20.3 microns, which yields an X-Y step between spots of 396 microns / 20.3 microns to give 19 spots. For example, a slide spotter a 384-well plate may be used, with up to about 18 384-well plates kept on a chilled plate to control evaporation. The samples "on deck" or queued in the plates may be of about 6,912. Slides on deck may be, e.g., forty, if six potter pins/shooters are used per robot arm. Basic functions or steps per cycle can include: clean, aspirate, prime/verify shooter, and spot. In operation, the present invention may be used in, e.g., expression analysis. Polymerase Chain Reaction Polymerase Chain Reaction (PCR) products, cDNAs, oligonucleotides and DNA fragments have been spotted on glass as high-density hybridization targets. Fluorescently labeled cDNAs derived from cellular extracts of mRNA have achieved a dynamic range (detection limit) of 1 in 10,000 to 100,000, allowing for detection of message in low and high abundance. Many experiments to measure differential expression have been reported for yeast, Arabidopsis and human DNAs. Presently, comprehensive and concise data on quantitative analysis of gene expression are available. Use of known expression data may be used to predict and measure known expression patterns having clinical/clinical research application with unknown samples to obtain real-time expression data.

For smaller arrays, a Hamilton 2200 automated pipeting robot is used to make arrays of oligonucleotide drops, ranging in size from about 100 nl to about 250 nl, with 1 mm spacing between dots. The small volumes of oligonucleotide solution used with the automated pipeting robot allows for rapid drying of the oligonucleotide drops. As with piezo-electric ink-jet printing methods, a Hamilton robot may be programmed to deliver nano to pico-liter size droplets with sub-millimeter spacing. Automated delivery of a oligonucleotide solution may use an ink-jet printing technique performed by, e.g., MicroFab (MicroFab Technologies, Inc., Piano, Tex.).

The present invention may be used with existing photochemical protocols and slide spotting technology, in conjunction with known expression levels for preselected and known genes, to optimize gene expression analysis using multiplexing of query samples by using a number of dyes and the full spectral imaging capabilities of the slide reader. The present invention may be used with slide readers or capacitance-coupled arrays to identify the expression levels of every gene of the entire organism at one time for multiple-multiplexed samples, in real time, with rapid turn-around and throughput, over time, and with consistency across samples.

Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Indeed, various modifications of the described compositions and modes of carrying out the invention which are obvious to those skilled in molecular biology or related arts are intended to be within the scope of the following claims.

Claims

What is claimed is:

1. A kit for oligonucleotide hybridization, the kit comprising: an array of nucleic acids immobilized on a solid support, wherein each of the immobilized nucleic acids includes a predetermined sequence; and a first labeled oligonucleotide complementary to the predetermined sequence of the immobilized nucleic acids, wherein the labeled oligonucleotide hybridizes with every nucleic acid on the array.

2. The kit of claim 1, wherein the first labeled oligonucleotide comprises a fluorescent label for detection.

3. The kit of claim 1, wherein the immobilized nucleic acids are the product of asymmetric amplification by the SMART^® polymerase chain reaction procedure and the first labeled oligonucleotide is complementary to a SMART^® primer oligonucleotide sequence.

4. The kit of claim 1, wherein the predetermined sequence comprises the T3 transcription initiation sequence.

5. The kit of claim 1, wherein the predetermined sequence comprises the T7 transcription initiation sequence .

6. The kit of claim 1, wherein the predetermined sequence comprises the Sp6 transcription initiation sequence .

7. The kit of claim 1, wherein the predetermined sequence comprises the M13 transcription initiation sequence .

8. The kit of claim 1, wherein the predetermined sequence comprises the consensus translation initiation sequence GCCA/GCCATGG.

9. The kit of claim 1, wherein the labeled oligonucleotide comprises a radioactive label.

10. The kit of claim 1, wherein the labeled oligonucleotide comprises a chemiluminescent label.

11. The kit of claim 1, wherein the solid support comprises glass.

12. The kit of claim 1, wherein the solid support comprises poly-L-lysine coated glass.

13. The kit of claim 1, wherein the solid support comprises polypropylene.

14. The kit of claim 2, wherein the fluorescent label is Cy3.

15. A kit for oligonucleotide hybridization, the kit comprising: one or more reagents to produce one or more nucleic acids, wherein at least one of the reagents further comprises an oligonucleotide having a predetermined sequence, the oligonucleotide being incorporated into each of the nucleic acids; a solid support for immobilizing the single stranded nucleic acids to provide an array of single stranded nucleic acids immobilized on the solid support; and a detectable oligonucleotide complementary to the predetermined sequence, whereby the detectable oligonucleotide hybridizes with every nucleic acid on the array.

16. The kit of claim 15, wherein the detectable oligonucleotide comprises a fluorescent label for detection.

17. The kit of claim 15, wherein the nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure and the detectable oligonucleotide is complementary to the predetermined sequence.

18. The kit of claim 15, wherein the predetermined sequence comprises the T3 transcription initiation sequence .

19. The kit of claim 15, wherein the predetermined sequence comprises the T7 transcription initiation sequence .

20. The kit of claim 15, wherein the predetermined sequence comprises the Sp6 transcription initiation sequence.

21. The kit of claim 15, wherein the predetermined sequence comprises the M13 transcription initiation sequence.

22. The kit of claim 15, wherein the predetermined sequence comprises the consensus translation initiation sequence GCC (A/G) CCATG.

23. The kit of claim 15, wherein the detectable oligonucleotide comprises a radioactive label for detection.

24. The kit of claim 15, wherein the detectable oligonucleotide comprises a chemiluminescent label for detection.

25. The kit of claim 15, wherein the solid support comprises glass.

26. The kit of claim 15, wherein the solid support comprises poly-L-lysine coated glass.

27. The kit of claim 15, wherein the solid support comprises polypropylene.

28. The kit of claim 15, wherein the reagents further comprise reagents for polymerase chain reaction amplification of double stranded nucleic acids, and wherein the predetermined sequence is used for asymmetric polymerase chain reaction, whereby one strand of the oligonucleotide is enriched to produce the single stranded predetermined sequence.

29. The kit of claim 15, wherein the reagents further comprise : reagents for polymerase chain reaction amplification of double stranded oligonucleotides; biotin end-labeled primer; and strepavidin beads, whereby the biotin labeled oligonucleotide strands are selected from denatured polymerase chain reaction product oligonucleotides by exposure of the denatured oligonucleotides to the strepavidin beads to produce the single stranded nucleic acids of the kit.

30. The kit of claim 16, wherein the detectable oligonucleotide comprises a Cy3 fluorescent label.

31. A system for oligonucleotide hybridization, the system comprising: an array of single stranded nucleic acids immobilized on a solid support, wherein the immobilized nucleic acids comprises a predetermined sequence; and a labeled oligonucleotide that hybridizes to the predetermined sequence present in each nucleic acid of the array to provide a quantitatively reproducible baseline signal for normalizing signals obtained from hybridization with the array.

32. The system of claim 31, wherein the labeled oligonucleotide comprises a fluorescent label.

33. The system of claim 31, wherein the nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure.

34. The system of claim 31, wherein the nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure and the predetermined oligonucleotide is complementary to the SMART^® primer oligonucleotide sequence.

35. The system of claim 31, wherein the predetermined sequence hybridizes with the T3 transcription initiation sequence.

36. The system of claim 31, wherein the predetermined sequence hybridizes with the T7 transcription initiation sequence.

37. The system of claim 31, wherein the predetermined sequence hybridizes with the Sp6 transcription initiation sequence.

38. The system of claim 31, wherein the predetermined sequence hybridizes with the M13 transcription initiation sequence.

39. The system of claim 31, wherein the predetermined sequence hybridizes with the consensus translation initiation sequence GCC (A/G) CCATGG.

40. The system of claim 31, wherein the reference oligonucleotide comprises a radioactive label.

41. The system of claim 31, wherein the reference oligonucleotide comprises a chemiluminescent label.

42. The system of claim 31, wherein the solid support comprises glass.

43. The system of claim 31, wherein the solid support comprises poly-L-lysine coated glass.

44. The system of claim 31, wherein the solid support comprises polypropylene.

45. The system of claim 32, wherein the fluorescent label is Cy3.

46. A system for oligonucleotide hybridization, the system comprising: one or more reagents to produce single stranded nucleic acids, wherein the reagents further comprise a primer oligonucleotide having a predetermined sequence for incorporation into each of the single stranded nucleic acids; a solid support for immobilizing the single stranded nucleic acids to provide an array of single stranded nucleic acids immobilized on the solid support; and a detectable oligonucleotide complementary to the predetermined sequence, whereby the detectable oligonucleotide hybridizes with every nucleic acod on the array and wherein the detectable oligonucleotide.

47. The system of claim 46, wherein the detectable oligonucleotide comprises a fluorescent label.

48. The system of claim 46, wherein the single stranded nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure.

49. The system of claim 46, wherein the single stranded nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure and the detectable oligonucleotide is complementary to a SMART^® primer oligonucleotide sequence.

50. The system of claim 47, wherein the primer oligonucleotide comprises the T3 transcription initiation sequence.

51. The system of claim 47, wherein the primer oligonucleotide comprises the T7 transcription initiation sequence.

52. The system of claim 47, wherein the primer oligonucleotide comprises the Sp6 transcription initiation sequence.

53. The system of claim 47, wherein the primer oligonucleotide comprises the M13 transcription initiation sequence.

54. The system of claim 47, wherein the primer oligonucleotide comprises the consensus translation initiation sequence GCC (A/G) CCATG.

55. The system of claim 47, wherein the detectable oligonucleotide comprises a radioactive label for detection.

56. The system of claim 47, wherein the detectable oligonucleotide comprises a chemiluminescent label for detection.

57. The system of claim 47, wherein the solid support comprises glass.

58. The system of claim 47, wherein the solid support comprises poly-L-lysine coated glass.

59. The system of claim 47, wherein the solid support comprises polypropylene.

60. The system of claim 47, wherein the reagents further comprise reagents for polymerase chain reaction amplification of nucleic acids, and the primer oligonucleotide used for asymmetric polymerase chain reaction, whereby at least one of the oligonucleotides used for amplification includes the predetermined sequence and is enriched to produce the single stranded nucleic acids.

61. The system of claim 47, wherein the reagents further comprise : reagents for polymerase chain reaction amplification of double stranded oligonucleotides; a biotin end-labeled primer oligonucleotide that includes the predetermined sequence; and a strepavidin beads, whereby the biotin labeled oligonucleotide strands are selected from denatured polymerase chain reaction products by exposure of the denatured polymerase chain reaction products to the strepavidin beads to produce the single stranded nucleic acids for binding to the solid support.

62. The system of claim 47, wherein the fluorescent label is Cy3.

63. A method for detecting a nucleic acid comprising the steps of: immobilizing one or more nucleic acids on a solid support, each of the one or more the single stranded nucleic acids including a predetermined sequence; and providing a first labeled oligonucleotide complementary to the predetermined sequence, whereby the first labeled oligonucleotide hybridizes with every nucleic acid on the solid support at the predetermined sequence to provide a quantitatively reproducible baseline signal for normalizing signals obtained from hybridization with the nucleic acids on the solid support.

64. The method of claim 63, further comprising the steps of: providing a second labeled oligonucleotide to provide a second signal; exposing the first labeled oligonucleotide and the second labeled oligonucleotide to the nucleic acids on the solid support under conditions permissive for hybridization; removing nonspecifically bound substances from the solid support; detecting the signal from the first label and the second label from the first and second labeled oligonucleotides hybridized to the nucleic acids on the solid support to obtain a ratio; and normalizing the ratio to the quantified signal of the first label.

65. The method of claim 63, wherein the first label and the second label are fluorescent.

66. The method of claim 63, wherein the nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure and the reference oligonucleotide is complementary to the SMART^® predetermined sequence.

67. The method of claim 63, wherein the predetermined sequence comprises the T3 transcription initiation sequence.

68. The method of claim 63, wherein the predetermined sequence comprises the T7 transcription initiation sequence.

69. The method of claim 63, wherein the predetermined sequence comprises the Sp6 transcription initiation sequence.

70. The method of claim 63, wherein the predetermined sequence comprises the M13 transcription initiation sequence.

71. The method of claim 63, wherein the predetermined sequence comprises the consensus translation initiation sequence GCC (A/G) CCATG.

72. The method of claim 63, wherein the first labeled oligonucleotide comprises a radioactive label.

73. The method of claim 63, wherein the first labeled oligonucleotide comprises a chemiluminescent label.

74. The method of claim 63, wherein the solid support comprises glass.

75. The method of claim 63, wherein the solid support comprises poly-L-lysine coated glass.

76. The method of claim 63, wherein the solid support comprises polypropylene.

77. The method of claim 64, wherein the first label is Cy₃ and the second label is Cys .

78. A method for hybridizing oligonucleotides, the method comprising: providing single stranded oligonucleotide probes; providing a solid support for immobilizing the single stranded oligonucleotide probes to provide an array of single stranded oligonucleotide probes immobilized on the solid support; immobilizing the single stranded oligonucleotide probes on the support; providing a reference oligonucleotide complementary to the primer oligonucleotide, wherein the reference oligonucleotide is labeled with a first label to provide a first signal; providing a test oligonucleotide labeled with a second label to provide a second signal; exposing the reference oligonucleotide and the test oligonucleotide to the array under conditions permissive for hybridization; removing nonspecifically bound substances from the solid support; quantitatively detecting the signal from the first label and the second label from oligonucleotides hybridized to the array to obtain a ratio; and normalizing the ratio to the quantified signal of the first label.

79. The method of claim 78, the method further comprising preparing the single stranded oligonucleotide probes by asymmetric polymerase chain reaction using only a single polymerase chain reaction primer, whereby one strand of the oligonucleotide is enriched.

80. The method of claim 78, the method further comprising preparing the single stranded oligonucleotide probes by polymerase chain reaction amplification of double stranded oligonucleotides, wherein the primer is end-labeled with biotin; the method still further comprising: heating the polymerase chain reaction product to denature double-stranded oligonucleotides; and selecting the biotin labeled oligonucleotide strand by exposure of the denatured oligonucleotides to strepavidin beads.

81. The method of claim 78, wherein the first label and the second label are fluorescent.

82. The method of claim 78, wherein the nucleic acids are the product of amplification by the SMART^® polymerase chain reaction procedure and the reference oligonucleotide is complementary to the SMART^® primer oligonucleotide sequence.

83. The method of claim 78, wherein the primer oligonucleotide comprises the T3 transcription initiation sequence.

84. The method of claim 78, wherein the primer oligonucleotide comprises the T7 transcription initiation sequence.

85. The method of claim 78, wherein the primer oligonucleotide comprises the Sp6 transcription initiation sequence.

86. The method of claim 78, wherein the primer oligonucleotide comprises the M13 transcription initiation sequence.

87. The method of claim 78, wherein the primer oligonucleotide comprises the consensus translation initiation sequence GCC (A/G) CCATG.

88. The method of claim 78, wherein the first and second labels are radioactive.

89. The method of claim 78, wherein the first and second labels are chemiluminescent.

90. The method of claim 78, wherein the solid support comprises glass.

91. The method of claim 78, wherein the solid support comprises poly-L-lysine coated glass.

92. The method of claim 78, wherein the solid support comprises polypropylene.

93. The method of claim 78, wherein the first label and the second label are fluorescent.

94. The method of claim 92, wherein the first label is Cy₃ and the second label is Cys .

95. The method of claim 78, wherein the reference oligonucleotide probe is chemically synthesized.

96. The method of claim 78, wherein the reference oligonucleotide probe is biologically synthesized.