Method of labeling nucleic acids

Abstract

The present invention relates to a method for labeling a nucleic acid characterized in that a ratio of signal intensities of each of labels of the labeled nucleic acids prepared from the same nucleic acid used as a template is substantially the same, irrelevant to the kinds of nucleic acids used as the template, a labeled nucleic acid prepared by the method, and a kit used for the method.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for preparing a labeled nucleic acid used in hybridization and a kit for the method, capable of performing gene expression analysis simply, quickly and at high reliability using a DNA chip or DNA microarray.

BACKGROUND ART

[0002] While all cells of an organism have a set of genes that are inherent in the organism, the kinds and amounts of genes expressed vary depending on the kinds of cells and cellular cycle. The patterns of the kinds and amounts off genes expressed in each cell or tissue are referred to as gene expression profile. There has been considered that the functions and characteristics of each cell are determined depending on the kinds and distributions of proteins existing in the cell at the time. There has been considered, therefore, that the functions and characteristics of the cell can be deduced from the analysis of the gene expression profile capable of measuring the amounts of synthesized proteins.

[0003] Also, it has been known that the gene expression profile may significantly change from that of a normal cell due to somatic change such as a disease. In other words, causal genes or genes that are used as diagnostic indices can be found from the analysis of the gene expression profile depending upon the disease or the like.

[0004] In recent years, there has been developed a DNA chip, a DNA microarray or the like capable of measuring simultaneously the expression of a large number of genes, wherein a large number of DNAs corresponding to a large number of genes are immobilized on a substrate made of glass or the like. Therefore, the gene expression profile can be measured.

[0005] The gene expression analysis using the above DNA chip or DNA microarray is usually performed by hybridization of a probe obtained by labeling cDNA with fluorescence, wherein the cDNA is derived from mRNA prepared from a cell to be analyzed, and a DNA chip or DNA microarray. In this analysis, there have been known (1) a single color method, wherein analysis is carried out using one kind of a fluorescent substance for labeling and two DNA chips or DNA microarrays which differ for each of the two kinds of samples to be compared, and (2) a dual color hybridization method (dual color method), wherein one DNA chip or DNA microarray is competitively hybridized with a probe obtained by labeling each cDNA with two kinds of fluorescent substances having different detection wavelengths, wherein the cDNA is derived from mRNA prepared from two kinds of cells to be compared.

[0006] The analysis according to the above single color method has a drawback that it is difficult to obtain an accurate gene expression ratio because there are some possibilities that the amount and state of DNA immobilized slightly vary for each of the DNA microarrays to be used, depending upon their preparation methods. In addition, there is a drawback that accurate comparison is difficult because the background intensities vary for each of the DNA chips and the DNA microarrays. Therefore, the analysis is generally carried out according to the dual color method.

[0007] The method for labeling a cDNA probe used in the above dual color method includes a direct labeling method [P. Hedge et al., BioTechniques, 29(3), 548-562 (2000)] and an indirect labeling method [D. D. Shoemaker et al., Nature, 409, 922-927 (2001)]. The direct labeling method, which is a method comprising reverse-transcribing mRNA, and incorporating a fluorescent substrate into cDNA during the cDNA synthesis, is simple. By contrast, the indirect labeling method is a labeling method comprising firstly synthesizing and purifying a non-labeled cDNA, and thereafter labeling the cDNA with a fluorescent dye by chemical reaction. An example of the above indirect labeling method includes a method comprising preparing a first-strand cDNA using a substrate having amino group during the reverse transcription of mRNA, and binding a fluorescent dye to the amino group. However, the indirect labeling method has some drawbacks in that procedures are complicated, that the time required for the preparation of the probe is long, and that purification procedures for cDNA need much steps, so that a final yield for the probe becomes low.

[0008] Therefore, there is currently most generally used a dual color method using a directly labeled cDNA probe [P. Hedge et al., BioTechniques, 29(3), 548-562 (2000)]. In the dual color method using the directly labeled cDNA probe, fluorescent substrates used for labeling are generally Cy3-labeled dUTP (or Cy3-labeled dCTP) and Cy5-labeled dUTP (or Cy5-labeled dCTP). The above direct labeling is carried out by reverse-transcribing mRNA with a reverse transcriptase in the copresence of four kinds of non-labeled substrates (dATP, dGTP, dCTP and dTTP) and Cy3-labeled dUTP (or Cy3-labeled dCTP) or Cy5-labeled dUTP (or Cy5-labeled dCTP), thereby synthesizing a first-strand cDNA.

[0009] There has been known that the length and the amount of the resulting first-strand cDNA in the above labeling vary depending on the fluorescent substrate used. For this reason, even if the mRNA of the same amount and the same molecular species exists in each sample, the fluorescent labeling ratio of the cDNA probe synthesized thereby (a ratio of the labeled signal intensity of Cy3-labeled nucleic acid to the labeled signal intensity of Cy5-labeled nucleic acid) differs for each gene. Therefore, the signal intensity ratio of the cDNA probes bound to a complementary fragment on the DNA chip or DNA microarray differs for each gene, thereby showing different apparent expression ratios. For instance, even if the same mRNA exists in each of two or more kinds of samples to be determined, when each mRNA is labeled with a different kind of a fluorescent substance, the ratio of the amounts of these fluorescent substrates incorporated into the labeled cDNA probe may differ for each gene. Therefore, the conventional dual color method using the directly labeled cDNA probe as mentioned above has a drawback that the ratio of the signal intensity ascribed to the labeling of the cDNA probe hybridized to a DNA on a DNA chip or DNA microarray differs for each gene, whereby showing different apparent expression ratios. In other words, if the dual color hybridization is carried out using a cDNA probe labeled with different labeled substrates using the same mRNA in the same amount, followed by correction analysis (for instance, global normalization), the expression of a case where the labeling is carried out with one labeling substrate and a case where the labeling is carried out with another labeling substrate is supposed to be substantially the same for all of the genes, but there are some cases where the genes not having substantially the same expression are generated.

[0010] Moreover, in order to carry out a method for preparing a directly labeled cDNA probe, there has been commercially available a kit comprising a reverse transcriptase so that a correction is made on the difference in incorporation ratio between different labeled substrates in the same nucleic acid used as a template. However, even if the above kit is used, there is a drawback that the ratio of the incorporation efficiencies of different labeled substrates, for instance, a ratio of the incorporation efficiency of a Cy3-labeled substrate to the incorporation efficiency of a Cy5-labeled substrate, cannot be made at the same level for all of the genes. Furthermore, according to the above kit, there are many cases where the ratio of the signal intensity of a signal ascribed to a Cy3-labeled nucleic acid to a signal ascribed to a Cy5-labeled nucleic acid, each labeled nucleic acid being prepared from the same nucleic acid used as a template, may fluctuate depending upon the kinds of nucleic acids used as the template. Therefore, the ratio of the labeled signal intensity after the subsequent global normalization treatment may not reflect the original abundance ratio of the nucleic acid as a template in some cases.

[0011] In the gene expression analysis using a DNA chip or DNA microarray, there are many cases where the expression alterations of 2 folds or more or ½ folds or less that of the control sample (control mRNA) is considered as a significant expression alteration. However, even if the same mRNA is used in the same amount, in the case of a labeling method involving a gene of which expression level is calculated to be 2 folds or more or ½ folds or less that of another gene, totally erroneous results are obtained so that it is difficult to obtain an accurate alteration of gene expression.

[0012] In addition, when a labeled substrate is incorporated into a nucleic acid in a general enzyme reaction, improvement of an incorporation efficiency has been tried by lowering a concentration of the non-labeled substrate and increasing a concentration of the labeled substrate, within a range in which the reaction ability of the enzyme can be maintained at a given level. In the labeling of a cDNA probe used in the dual color hybridization method, the concentrations of the non-labeled substrate and the labeled substrate have been also taken into consideration in order to increase the incorporation of the labeled substrate into the nucleic acid as described above. However, the above-mentioned drawbacks inherently owned by the dual color hybridization method have not been taken into consideration in the current situation.

[0013] Therefore, there has been desired a method for labeling a target nucleic acid capable of understanding the behavior of an accurate gene expression in the gene expression analysis.

DISCLOSURE OF INVENTION

[0014] An object of the present invention is to provide a method for labeling a nucleic acid characterized in that a ratio of signal intensities of each of labels of the labeled nucleic acids prepared from the same nucleic acid used as a template is substantially the same, irrelevant to the kinds of nucleic acids used as the template, a labeled nucleic acid prepared by the method, and a kit for the method.

[0015] A first invention of the present invention relates to a method for labeling each of nucleic acids in a nucleic acid sample containing plural nucleic acids with at least two kinds of different labeled substances distinguishable from each other, characterized in that a ratio of signal intensities of each of labels of the labeled nucleic acids is substantially the same, irrelevant to the kinds of nucleic acids used as the template, when the labeled nucleic acids are prepared from arbitrary nucleic acids as a template, and the labeled nucleic acids are labeled with the labeled substrate. More concretely, the first invention relates to a method for labeling a nucleic acid, wherein the method is a method for labeling the nucleic acid with at least two kinds of different labeled substances distinguishable from each other, and wherein the method comprises the step of labeling the nucleic acid in a nucleic acid sample containing plural kinds of nucleic acids by use of:

[0016] one labeled substrate which is labeled with a labeling substance and a non-labeled substrate corresponding thereto, in an amount ratio satisfying the following conditions that a ratio of:

[0017] a) a signal intensity of a signal ascribed to a labeled nucleic acid prepared with the nucleic acid in the nucleic acid samples as a template, wherein the labeled nucleic acid is labeled with the labeled substrate, to

[0018] b) a signal intensity of a signal ascribed to a labeled nucleic acid prepared with the same nucleic acid as that of the above item a) as a template, wherein the labeled nucleic acid is labeled with a labeled substrate different from the labeled substrate of the above item a)

[0019] in each of the nucleic acids in the nucleic acid sample is substantially the same, irrelevant to the kinds of the nucleic acids to be used as a template.

[0020] In the first invention of the present invention, it is desired that a labeled nucleic acid may be preferably prepared by reverse transcription reaction from the nucleic acid used as a template, and that the different labeled substrate is preferably a Cy3-labeled substrate or a Cy5-labeled substrate. There can be preferably used a reaction mixture used for labeling comprising the non-labeled substrate and the Cy3-labeled substrate, wherein its ratio within the range of from 1:1 to 5:1, and/or a reaction mixture used for labeling comprising the non-labeled substrate and the Cy5-labeled substrate, wherein its ratio within the range of from 3:1 to 10:1. In other words, in the first invention of the present invention, it is desired that the nucleic acids in the nucleic acid sample are labeled in a reaction mixture containing the non-labeled substrate and the Cy3-labeled substrate preferably in a concentration ratio (the non-labeled substrate/the Cy3-labeled substrate) within the range of from 1/1 to 5/1. In addition, it is desired that the nucleic acids in the nucleic acid sample are labeled in a reaction mixture containing the non-labeled substrate and the Cy5-labeled substrate in a concentration ratio (the non-labeled substrate/the Cy5-labeled substrate) within the range of from 3/1 to 10/1.

[0021] A second invention of the present invention relates to a labeled nucleic acid prepared by the method for labeling a nucleic acid of the first invention of the present invention.

[0022] A third invention of the present invention relates to a kit for labeling a nucleic acid comprising an instruction manual describing the method for labeling a nucleic acid of the first invention of the present invention, i.e. a kit comprising an instruction manual describing the procedures of the method for labeling a nucleic acid of the first invention of the present invention.

[0023] In the third invention of the present invention, there can be preferably used a kit comprising an instruction manual describing a method for preparing a mixed substrate by use of the non-labeled substrate and the Cy3-labeled substrate, wherein the concentration ratio thereof (the non-labeled substrate/the Cy3-labeled substrate) ranges from 1/1 to 5/1; and/or a kit comprising an instruction manual describing a method for preparing a mixed substrate by use of the non-labeled substrate and the Cy5-labeled substrate, wherein the concentration ratio thereof (the non-labeled substrate/the Cy5-labeled substrate) ranges from 3/1 to 10/1.

[0024] Furthermore, a fourth invention of the present invention relates to a kit for labeling a nucleic acid, comprising:

[0025] (1) a reaction vessel containing a reaction mixture comprising a Cy3-labeled substrate and a non-labeled substrate corresponding thereto in a concentration ratio (non-labeled substrate/Cy3-labeled substrate) of from 1/1 to 5/1, and comprises a non-labeled nucleotide substrate other than the above non-labeled substrate, and/or

[0026] (2) a reaction vessel containing a reaction mixture comprising a Cy5-labeled substrate and a non-labeled substrate corresponding thereto in a concentration ratio (non-labeled substrate/Cy5-labeled substrate) within the range of from 3/1 to 10/1, and comprises a non-labeled nucleotide substrate other than the above non-labeled substrate, wherein the reaction mixture is a reaction mixture for one-time use or defined times of use. The above kit for labeling a nucleic acid may further comprise a reverse transcriptase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a diagram showing “Scatter Plot” of the fluorescent intensity in a case where a fluorescent-labeled probe prepared by the method of the present invention is used.

[0028] FIG. 2 is a diagram showing “Scatter Plot” of the fluorescent intensity in a case where a fluorescent-labeled probe prepared by a conventional method is used.

[0029] FIG. 3 is a diagram showing “Scatter Plot” of the fluorescent intensity in a case where a fluorescent-labeled probe prepared with a commercially available kit is used.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] The present inventors have found that labeling of a target nucleic acid, capable of understanding the behavior of gene expression surprisingly accurately in gene expression analysis, can be achieved by setting a concentration ratio of a non-labeled substrate to a labeled substrate at a particular ratio for each of at least two kinds of labeled substrates, for instance, fluorescent substrates. They have further found that according to the labeling method based on the concentration ratio, a kit for fluorescent-labeling a probe capable of performing accurate gene expression analysis using the dual color hybridization method in high accuracy, whereby the present inventors have achieved the present invention.

[0031] In the present specification, the evaluation of the method for labeling a nucleic acid is carried out by, for instance, the steps of:

[0032] (1) carrying out a reverse transcription reaction using at least two kinds of different labeled substrates with the same amount of mRNA as a template for each of the labeled substrates to give a labeled cDNA,

[0033] (2) carrying out hybridization using each of the labeled cDNAs obtained in the above step (1) and a DNA chip or DNA microarray, and

[0034] (3) calculating an extent of variance of the ratios of signal intensities between the signals ascribed to each of the labeled substrates for spots showing significant signal intensity for all of the labeled substrates by correction analysis of a signal ascribed to the labeled substrate of each complex, wherein the complex is formed by the hybridization in the above step (2) (a complex of the labeled cDNA with a DNA which is immobilized on the DNA chip or DNA microarray).

[0035] Concretely, for instance, the method for labeling a nucleic acid of the present invention can be evaluated by using as an index an extent of variance of a ratio of signal intensities of the spots showing significant signal intensity for both Cy3 and Cy5, when the same mRNA in the same amount is used for labeling with different labeled substrates, for instance, fluorescent substrates, concretely CyDye-labeled substrates (Cy3-labeled substrate and Cy5-labeled substrate), dual color hybridization is carried out, and thereafter a signal intensity ascribed to Cy5 is subjected to correction analysis against to a signal intensity ascribed to Cy3.

[0036] In the present specification, the above extent of variance refers to a value calculated by [standard deviation of (logarithmic value of Cy3/Cy5 ratio)×2.5].

[0037] In addition, in the above evaluation, when the above value showing the extent of variance is 2 or less, it is shown that the labeling method is a labeling method capable of understanding accurately the behavior of gene expression.

[0038] In the present specification, “substantially the same” refers to the fact that in the scatter plot of signal intensity of label ascribed to each of the labeled nucleic acids labeled with labeling substances distinguishable from each other, the signal intensity of the signal ascribed to arbitrary one labeling substance/the signal intensity of the signal ascribed to another labeling substance falls within the range of 1/2 to 2/1.

[0039] In the present specification, the “signal intensity” refers to a value obtained by subjecting a DNA chip or DNA microarray to a fluorescence reader (array scanner) to measure the spots showing fluorescence, wherein DNA chip or DNA microarray has been hybridized with labeled nucleic acids (probes), thereafter washed and dried, and subtracting an average intensity of the background signals in the surroundings of each spot from an average intensity of the fluorescence signals showing the spots which are immobilized regions of the nucleic acids on a DNA chip or DNA microarray, when the fluorescence signal intensity of each spot is determined with a quantitative image analytical software program.

[0040] In the present specification, the “significant signal intensity” refers to a case where [Mean of a given one spot signal] shows a value greater than [Mean+2×SD (standard deviation) of the background signal] of the surroundings of each spot.

[0041] In the present specification, the correction of a signal intensity of a signal ascribed to a labeling substance, for instance, a signal intensity of a signal ascribed to a fluorescent substance, concretely a signal intensity of a signal ascribed to Cy3 and a signal intensity of a signal ascribed to Cy5, can be carried out by using global normalization method. Concretely, for instance, the signal intensity ascribed to Cy5 is corrected so that logarithmic values of the ratio of the signal intensity of the signal ascribed Cy3/the signal intensity of the signal ascribed to Cy5 has an average value of 0, wherein the values are taken for each of the genes having a significant signal intensity in the sample to be measured.

[0042] In the present specification, the “extent of variance” uses as an index a value calculated by a constant obtained by [standard deviation of (logarithmic value of Cy3/Cy5 ratio)×2.5], when a histogram of the logarithmic value of the Cy3/Cy5 ratio for the spots showing significant signal intensity for labeling substances, for instance, fluorescent substances, concretely for both Cy3 and Cy5, is drawn, wherein the histogram shows a nearly normal distribution.

[0043] The method for calculating the index for the above “extent of variance” utilizes the fact that 99% of numerical values fall within 2.5 standard deviations from the mean, when it is supposed that the data show a normal distribution. The index represents the variance from the mean in which the great majority of spots are distributed excluding few exceptional spots.

[0044] In the present specification, the “labeled substrate” refers to a substance in which a labeling substance, for instance, a fluorescent substance, a radioactive compound, biotin, amino group or the like is added to a nucleotide substrate, and includes concretely Cy5-dUTP, Cy5-dCTP, Cy3-dUTP, and Cy3-dCTP.

[0045] The above “nucleotide substrate” includes a substrate usable in nucleic acid synthesis, concretely dATP, dGTP, dCTP, dTTP, dUTP or the like. In the present specification, the representation “nucleotide substrate” means a non-labeled substrate unless specified otherwise.

[0046] In the present specification, the phrase “non-labeled substrate corresponding to a labeled substrate” or the term “non-labeled substrate” refers to a nucleotide substrate which is incorporated in place of the above labeled substrate. There are included, for instance, but not particularly limited to, dTTP when a labeled substrate is a labeled dUTP or the like, concretely, for instance, Cy3-dUTP or Cy5-dUTP; and dCTP when a labeled substrate is a labeled dCTP or the like, concretely, for instance, Cy3-dCTP or Cy5-dCTP.

[0047] In the present specification, “plural” in the phrase “nucleic acid sample containing plural kinds of nucleic acids” has the same definition as two kinds or more.

[0048] The present invention will be hereinafter described in detail.

[0049] (1) Method for Labeling Nucleic Acid of the Present Invention and Labeled Nucleic Acids Prepared by the Method

[0050] The method for labeling a nucleic acid of the present invention is a method for labeling each of nucleic acids in a nucleic acid sample containing plural kinds of nucleic acids with two or more different labeled substances distinguishable from each other, wherein one of the significant features of the method resides in that a ratio of each of labeled signal intensities of the labeled nucleic acids is substantially the same, irrelevant to the kinds of nucleic acids used as the template, wherein the labeled nucleic acids are those prepared from arbitrary nucleic acids as a template, and labeled with the labeled substrate. In other words, the labeling method of the present invention is a method for labeling a nucleic acid with at least two kinds of different labeling substances distinguishable from each other, wherein one of the features of the method resides in that there is carried out the step of labeling the nucleic acid (concretely all nucleic acids or a part of nucleic acids) in a nucleic acid sample containing plural kinds of nucleic acids are labeled by use of:

[0051] one labeled substrate which is labeled with a labeling substance and

[0052] a non-labeled substrate corresponding thereto,

[0053] in an amount ratio satisfying the following conditions that a ratio of:

[0054] a) a signal intensity of a signal ascribed to a labeled nucleic acid prepared with the nucleic acid in the nucleic acid samples as a template, wherein the labeled nucleic acid is labeled with the labeled substrate, to

[0055] b) a signal intensity of a signal ascribed to a labeled nucleic acid prepared with the same nucleic acid as that of the above a) as a template, wherein the labeled nucleic acid is labeled with a labeled substrate different from the labeled substrate of the above a)

[0056] in each of the nucleic acids in the nucleic acid sample is substantially the same, irrelevant to the kinds of the nucleic acids to be used as a template.

[0057] According to the labeling method of the present invention, since the non-labeled substrate and the labeled substrate are used at the above content ratio, in a case where the same mRNA is used as a template, there is exhibited an excellent effect that substantially the same expression ratio (Cy3 signal/Cy5 signal ratio) can be obtained, irrelevant to the kinds of genes. In addition, according to the labeling method of the present invention, since the non-labeled substrate and the labeled substrate are used at the above content ratio, there is exhibited an excellent effect that the behavior of gene expression can be understood accurately.

[0058] According to the method for labeling a nucleic acid of the present invention, the nucleic acid to be used as a template can be prepared from a sample possibly containing a nucleic acid such as DNA or RNA. The above samples include, for instance, but are not particularly limited to, biological samples such as whole blood, serum, buffy coat, urine, feces, cerebrospinal fluid, semen, saliva, tissues (for instance, cancer tissue, lymph nodes and the like), and cell cultures (for instance, mammalian cell cultures, bacterial cultures and the like); nucleic acid-containing samples such as of viroids, viruses, bacteria, fungi, yeast, plants, and animals; samples possibly contaminated or infected with microorganisms such as viruses or bacteria (foods, biological products, and the like); or samples possibly containing organisms, such as soil and wastewater. The samples include samples obtained by appropriately treating each of the biological samples, the nucleic acid-containing samples, the samples potentially contaminated or infected with microorganisms and the samples possibly containing organisms mentioned above.

[0059] As the above “nucleic acid as a template,” any of RNAs and DNAs can be preferably used. When the analysis of gene expression is carried out in a cell, mRNA obtained from the cell can be used as the above “nucleic acid as a template.”

[0060] The method for labeling a nucleic acid of the present invention can be applied to any of nucleic acids, as long as the nucleic acids are nucleic acids capable of labeling with a commonly used labeling substance. The method for labeling a nucleic acid of the present invention can, for instance, be used, but not particularly limited to, during the first strand cDNA synthesis reaction (reverse transcription reaction).

[0061] The reverse transcriptase which can be used in the above reverse transcription reaction includes, for instance, but is not particularly limited to, AMV RTase, MMLV RTase, RAV-2 RTase and the like.

[0062] In addition, in the labeling method of the present invention, when a CyDye-labeled substrate, for instance, is used, it is desirable that a final concentration of the CyDye-labeled substrate in a reaction mixture is, but not particularly limited to, preferably within the range of 0.02 mM to 0.3 mM, more preferably within the range of 0.025 mM to 0.1 mM, from the viewpoint of obtaining an excellent economic advantage and high operability, concretely, for instance, from the viewpoint of easily completely removing an unreacted CyDye-labeled substrate from a solution containing a labeled nucleic acid in the purification step, thereby giving an excellent background, and from the viewpoint of incorporating a labeled substrate into a nucleic acid in an amount sufficient for the enzyme reaction, thereby improving a signal intensity of the resulting labeled nucleic acids.

[0063] The method for labeling a nucleic acid of the present invention can be carried out by optimizing a concentration ratio of the non-labeled substrate to the labeled substrate used during the fluorescence-labeled cDNA probe synthesis reaction using mRNA (i.e., reverse transcription reaction). The ratio for the above non-labeled substrate/labeled substrate is expressed as, for instance, a concentration ratio of CyDye-labeled dUTP/dTTP in the use of CyDye-labeled dUTP, or as a concentration ratio of CyDye-labeled dCTP/dCTP in the use of CyDye-labeled dCTP.

[0064] In the labeling method of the present invention, it is also desirable that the amounts of the nucleotide substrates used for nucleic acid synthesis, concretely dATP, dGTP, dCTP, and dTTP (including dUTP) are preferably of equivalence. Concretely, it is desirable that the total amount thereof (concentration) are of equivalence between a case of the labeled substrates used and corresponding non-labeled substrates thereof, and a case of other nucleotide substrates. In a embodiment of the present invention, in a case where, for instance, the CyDye-labeled dUTP is used at a concentration of 0.05 mM in a reaction mixture, it is preferable, but not particularly limited to, that dTTP is used at a concentration of 0.1 mM in a reaction mixture (a combined concentration of the labeled dUTP and dTTP being 0.15 mM), and that each of dATP, dGTP and dCTP is used at a concentration of 0.15 mM in the reaction mixture when the ratio of the non-labeled substrate/labeled substrate is 2.

[0065] As described above, the labeling method of the present invention can be evaluated by determining whether or not there is a difference in a variance of signal intensity ratio of each spot, or a ratio of spots each having a significant signal, due to the difference in the kind of a fluorescent dye Cy3 or Cy5 used for labeling, when the labeled probe is prepared by varying a ratio of the non-labeled substrate/labeled substrate with fixing a concentration of the CyDye-labeled substrate in the above concentration range.

[0066] The above “ratio of spots having a significant signal” is used as an index for the signal intensity. Here, the larger the number of the significant spots is, the larger the ratio of spots having a significant signal intensity against the surrounding background, and the data for expression alterations (expression profile) are obtained with greater significance for a larger number of genes, whereby making it preferable for the analysis.

[0067] Furthermore, the labeling method of the present invention can be evaluated for each of the Cy3-labeled substrate and the Cy5-labeled substrate from the viewpoint of evenness in the signal intensity ratio for each of the genes, by preparing labeled nucleic acids (probes) with varying a ratio of the non-labeled substrate/labeled substrate against a given concentration of the Cy3-labeled substrate and Cy5-labeled substrate, and carrying out dual color hybridization analysis using all ratios for each substrate.

[0068] In the labeling method of the present invention, it is desirable, for instance, but not particularly limited to, that in a case where the same mRNA is used as a template, the range for the concentration ratio of the non-labeled substrate/labeled substrate is preferably 1/1 to 5/1, especially preferably 1.5/1 to 4/1, in a Cy3-labeled substrate, and that the range is preferably 3/1 to 10/1, especially preferably 4.5/1 to 9/1, for a Cy5-labeled substrate, from the viewpoint of obtaining substantially the same expression ratio (ratio of Cy3 signal/Cy5 signal), irrelevant to the kinds of the genes.

[0069] According to the labeling method of the present invention, there is exhibited an excellent effect that there can be prepared a labeled nucleic acid having an abundance ratio inherently owned by the nucleic acid used as a template in a nucleic acid-containing sample, for instance, but not particularly limited to, an abundance ratio of mRNA. Therefore, labeled nucleic acids prepared by the labeling method of the present invention are also encompassed in the present invention.

[0070] The labeled nucleic acid of the present invention can be utilized for all of the methods, as long as the hybridization is carried out according to the dual color method. For instance, since a nucleic acid labeled by the labeling method of the present invention has an inherent abundance ratio between two kinds of samples in the nucleic acid-containing sample, there can be preferably used in, for instance, but not particularly limited to, a hybridization method using a DNA micro array.

[0071] In addition, according to the labeling method of the present invention, the accuracy of the analysis of expressed genes can be improved in gene expression analysis according to the dual color hybridization method because expression alterations of 1.5 folds can be judged to be a significant alternation at a probability of about 99%, in contrast to alterations of 2 folds or more have been judged to be a significant difference in expression in the conventional method.

[0072] The labeled nucleic acid of the present invention is a nucleic acid used as a template in a nucleic acid sample containing plural kinds of nucleic acids, for instance, but not particularly limited to, a labeled nucleic acid having an abundance ratio of mRNA. Further, a feature of the labeled nucleic acid of the present invention resides in that the signal intensity ratio of each signal ascribed to labeled nucleic acids prepared from a given nucleic acid as a template is substantially the same, wherein the labeled nucleic acids have different labeled substrates, irrelevant to the kinds of the nucleic acids used as a template.

[0073] Furthermore, by using the labeling method of the present invention, the number of genes which can be analyzed at high reliability can be increased under the conditions that give as large a ratio of the significant spots as possible. In the present invention, the accuracy of gene expression analysis using the DNA chip or DNA microarray is increased so that expression alterations of within 2 folds can be also judged as a significant difference.

[0074] Therefore, according to the labeling method of the present invention, there is provided a method for analyzing gene profile characterized by the use of the labeled nucleic acid obtained by the labeling method of the present invention.

[0075] (2) Kit for Labeling Nucleic Acid of the Present Invention

[0076] The present invention provides a kit for labeling a nucleic acid used in the method for labeling a nucleic acid of the present invention described above.

[0077] In one embodiment of the present invention, the kit for labeling a nucleic acid of the present invention includes a kit comprising an instruction manual describing procedures for the labeling method of the present invention in a packaged form. In a preferred embodiment, the kit for labeling a nucleic acid of the present invention resides has a feature that the kit comprises an instruction manual describing a method for preparing a mixed substrate containing the Cy3-labeled substrate and the non-labeled substrate corresponding thereto in a concentration ratio (the non-labeled substrate/the Cy3-labeled substrate) within the range of from 1/1 to 5/1, preferably 1.5/1 to 4/1, and/or an instruction manual describing a method for preparing a mixed substrate containing the Cy5-labeled substrate and the non-labeled substrate corresponding thereto in a concentration ratio (the non-labeled substrate/the Cy5-labeled substrate) within the range of from 3/1 to 10/1, preferably 4.5/1 to 9/1. In addition, the kit of the present invention may comprise a non-labeled substrate and/or a labeled substrate. Furthermore, the kit of the present invention may comprise various necessary reagents including a reverse transcriptase, a reverse transcription reaction buffer and the like. Alternatively, a commercially available enzyme having reverse transcription activity may be selected and used in accordance with the instruction manual. The reverse transcriptase is not particularly limited, and AMV RTase, MMLV RTase, or RAV-2 RTase can be preferably used.

[0078] The above “instruction manual” refers to a printed matter describing a method for using the kit, for instance, a method for preparing a reverse transcription reaction reagent solution, the mixing ratio of the non-labeled substrate to the labeled substrate and their amounts, recommended reaction conditions or the like. The instruction manual includes, in addition to instruction manuals in the form of a pamphlet or leaflet, labels attached to a kit and description given on the package housing the kit. Furthermore, there is included information disclosed or provided via electronic media such as internet.

[0079] Furthermore, the kit used for a method for detecting a target nucleic acid may be a kit comprising, in addition to the above instruction manual and the reverse transcription reaction reagent, an oligonucleotide primer for the reverse transcription reaction (random primer or oligo-dT primer) or the like. The kit may further comprise a membrane filter unit for purifying the resulting labeled nucleic acid.

[0080] According to the kit for labeling a nucleic acid of the present invention, a labeled probe capable of carrying out analysis at high accuracy analysis can be prepared simply and under conditions in which the amount of CyDye-labeled substrate used is reduced.

[0081] Further, another embodiment of the kit for labeling a nucleic acid of the present invention includes a kit comprising:

[0082] (1) a reaction vessel containing a reaction mixture in an amount of one-time use or defined times of use, wherein the reaction mixture comprises a Cy3-labeled substrate and a non-labeled substrate corresponding thereto in a concentration ratio (non-labeled substrate/Cy3-labeled substrate) of from 1/1 to 5/1, preferably from 1.5/1 to 4/1, and comprises a non-labeled nucleotide substrate other than the above non-labeled substrate, and/or

[0083] (2) a reaction vessel containing a reaction mixture in an amount for one-time use or defined times of use, wherein the reaction mixture comprises a Cy5-labeled substrate and a non-labeled substrate corresponding thereto in a concentration ratio (non-labeled substrate/Cy5-labeled substrate) within the range of from 3/1 to 10/1, preferably from 4.5/1 to 9/1, and comprises a non-labeled nucleotide substrate other than the above non-labeled substrate. The kit may further comprise the above instruction manual, a reverse transcriptase, reagents for reverse transcription reaction, an oligonucleotide primer for the reverse transcription reaction (random primer or oligo-dT primer), a gel filtration column for purifying the labeled nucleic acid obtained, and the like. Here, the above “reaction mixture in an amount for one-time use or defined times of use” refers to a reaction mixture in an amount suitable for carrying out the reaction once or previously determined number of times.

[0084] In the kit of the present invention, the reaction mixture may be dispensed to a single reaction vessel for one-time use (referred to as one-time reaction vessel), or may be dispensed to a single reaction vessel for the defined times of use (referred to as multiple reaction vessel). According to the above one-time reaction vessel, the labeling method of the present invention can be carried out conveniently by allowing a user to add the target nucleic acid to be labeled in a given amount as instructed according to the instruction manual or the like, and subjecting the reaction vessel under the instructed reaction conditions. According to the multiple reaction vessel, the labeling method of the present invention can be carried out conveniently by dispensing the reaction mixture in a given amount as instructed in the instruction manual or the like and the target nucleic acid to be labeled into separate reaction vessels, and subjecting the reaction vessels under the instructed reaction conditions.

[0085] The above reaction vessel includes, for instance, a 1.5 ml-capacity mini-tube, a 200 μl-capacity micro-tube, and the like, and the volume of the vessel is not limited to those exemplified above.

[0086] The present invention will be described in more detail hereinbelow by means of Examples, without intending to limit the present invention to the scope of the Examples.

EXAMPLE 1

[0087] Various concentration ratios were set for a Cy3- or Cy5-labeled substrate (Cy3-dUTP or Cy5-dUTP) and a non-labeled substrate (dTTP). Each of the substrate concentrations is shown in Table 1.

[0088] Using each substrate so as to have the substrate concentrations shown in Table 1 in each of the Cy3 and Cy5 systems, the same mRNA was used in the same amount of mRNA for labeling the mRNA, to give each of labeled cDNA probes (Cy3-labeled cDNA probe and Cy5-labeled cDNA probe). In the gene expression analysis using the labeled cDNA probes obtained and a DNA chip or DNA microarray, studies on optimization of the above concentration ratio were conducted so that a ratio of a Cy3 fluorescence signal intensity to a Cy5 fluorescence signal intensity is substantially the same for all the genes.

[0089] Concretely, the concentration ratios of the non-labeled substrate/labeled substrate were set at 5/1, 3.5/1 and 2/1 to determine the concentration ratio at which analytical results with high accuracy could be obtained.

TABLE 1
Non-Labeled Substrate/Labeled Substrate = 5/1
dATP    0.30 mM
dGTP    0.30 mM
dCTP    0.30 mM
dTTP    0.25 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 3.5/1
dATP    0.225 mM
dGTP    0.225 mM
dCTP    0.225 mM
dTTP    0.175 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 2/1
dATP    0.15 mM
dGTP    0.15 mM
dCTP    0.15 mM
dTTP    0.10 mM
Cy-dUTP 0.05 mM

[0090] The preparation of the labeled cDNA probes were carried out as follows.

[0091] First, polyA(+) RNA was prepared from human HL-60 cells using Triazol Reagent (manufactured by GIBCO BRL) and Oligotex-dT30<Super> (manufactured by Takara Bio Inc.) in accordance with the protocol for each of the kits. Each of a Cy3-labeled cDNA probe and a Cy5-labeled cDNA probe was prepared and purified using RNA Fluorescence Labeling Core Kit (M-MLV Version) (manufactured by Takara Bio Inc.) in accordance with the protocol for the kit, with 1 μg of the polyA(+) RNA obtained as a template. Here, each of the substrates of the above concentration was used in place of a 10×dNTP Mixture contained in the above kit.

[0092] Next, hybridization between IntelliGene™ Human 1K Set I (manufactured by Takara Bio Inc.) and the above labeled cDNA probes was carried out in accordance with the instruction manual for IntelliGene™ M (manufactured by Takara Bio Inc.). Thereafter, the DNA chip or DNA microarray after the hybridization was washed and dried. Subsequently, the DNA chip or DNA microarray was scanned using Affymetrix 428 Array Scanner (manufactured by Affymetrix) to obtain fluorescent images for each of Cy3 (excitation wavelength: 532 nm, detection wavelength: 570 nm) and Cy5 (excitation wavelength: 635 nm, detection wavelength: 670 nm).

[0093] Next, the signal intensity of each spot on the fluorescent images obtained was calculated using quantitative image analytical software program, ImaGene 4.0 (manufactured by BioDiscovery). The ratio of Cy3/Cy5 signal intensity for each spot was expressed as a logarithmic value, and the correction of the signal intensities between Cy3 and Cy5 was carried out by global normalization method, by which correction was made so that an average signal intensity ratio would be 0 for all spots.

[0094] The expression ratio (ratio of Cy3/Cy5 signal intensity) was calculated from the corrected signal intensity ratio, and the range of the expression ratio for 99% convergence in distribution of the spots that are significant signals for both Cy3 and Cy5 was determined. The above range of the expression ratio was used as an index of analytical accuracy. Here, those of which Mean (average value) of the spot signals shows a value greater than the value of Mean+2×SD (standard deviation) of the background signal of the surrounding of each spot are defined as significant signals.

[0095] The concentration ratio of the non-labeled substrate/labeled substrate, the range of the expression ratio for 99% convergence in distribution of the significant spots at the concentration ratio, and the number of significant spots (%) for each of Cy3 and Cy5 are shown in Table 2.

TABLE 2
Non-Labeled Substrate/	Expression	Effective Spot	Effective Spot
Labeled Substrate	Ratio	for Cy3	for Cy5
5/1	1/2.12-2.12	38%	43%
3.5/1	1/2.40-2.40	45%	47%
2/1	1/2.86-2.86	53%	47%

[0096] As shown in Table 2, it was found that since the distribution of the expression ratio more closely converges to 1 when the concentration ratio of non-labeled substrate/labeled substrate is made higher (non-labeled substrate/labeled substrate=5/1) as compared to that when the concentration ratio of the non-labeled substrate/labeled substrate is made lower (non-labeled substrate/labeled substrate=2/1), the accuracy is increased.

EXAMPLE 2

[0097] (1) Studies on Combinations of Concentration Ratios of Non-Labeled Substrate/Labeled Substrate

[0098] By setting the concentration ratio of the non-labeled substrate/labeled substrate high (5/1) or low (2/1), studies were conducted on which of the concentration ratios was suitable for each of Cy3 and Cy5. Here, the preparation of labeled cDNA probes, hybridization with a DNA microarray, washing, scanning and analysis were carried out under the same conditions as those described in Example 1 except that the concentration ratios of the non-labeled substrate/labeled substrate were changed. As the DNA microarray, there was used IntelliGene™ Human Cancer Chip Ver. 2.0 (manufactured by Takara Bio Inc.). The combinations studied and the results of the range of the expression ratio are shown in Table 3.

	TABLE 3
	Non-Labeled Substrate/		Cy5
	Labeled Substrate		5/1	2/1
	Cy3	5/1	1.96(i)	2.99(iii)
		2/1	1.80(ii)	2.27(iv)

[0099] In Table 3, (iv) shows a comparative example by a conventional method [P. Hedge et al., BioTechniques, 29(3), 548-562 (2000)].

[0100] The “Scatter Plot” in a case where the concentration ratio of the non-labeled substrate/labeled substrate for Cy3 was 2/1, and where the concentration ratio of the non-labeled substrate/labeled substrate for Cy5 was 5/1 [(ii) in Table 3] is shown in FIG. 1. The “Scatter Plot” in a case where the concentration ratio of the non-labeled substrate/labeled substrate for Cy3 was 2/1, and where the concentration ratio of the non-labeled substrate/labeled substrate for Cy5 was 2/1 [(iv) in Table 3] is shown in FIG. 2.

[0101] FIGS. 1 and 2 are diagrams each showing signal intensities obtained when each fluorescent probe was used. In the figures, the X-axis is a Cy3 signal intensity, and the Y-axis is a Cy5 signal intensity, wherein an open circle (O) represents a spot exhibiting significant signal intensity; and a cross (+) represents a spot exhibiting non-significant signal intensity. Also, in the figures, a solid line is a theoretical line in which the expression ratio of the Cy3 signal intensity to the Cy5 signal intensity becomes 1:1, a broken line is a theoretical line in which the expression ratio of the Cy3 signal intensity to the Cy5 signal intensity becomes 2:1 or 1:2; and each dotted line is the theoretical line in which the expression ratio of the Cy3 signal intensity to the Cy5 signal intensity becomes 3:1 or 1:3.

[0102] As a result, as to the expression ratio of the Cy3 signal intensity to the Cy5 signal intensity, the expression ratio is supposed to be 1:1 for all of the genes. However, as shown in FIG. 2, the range of the Cy3/Cy5 ratio deduced to give a 99% convergence in distribution of significant signal spots was calculated to be 1/2.27 to 2.27 based on the (standard deviation of ratio of the Cy3/Cy5 signal intensity). In consideration of the significant difference of expression alteration of ½ folds or less or 2 folds or more as generally said, there is a risk that false-positive data may be contained that lead to an erroneous interpretation.

[0103] It was found, however, that the variance of spots is notably smaller in FIG. 1 as compared to that of FIG. 2. In other words, it was found that the labeling method shown in (ii) of Table 3 (FIG. 1) was found be a method capable of causing a smaller variance (uniform) of the ratio of labeling signal intensity due to the difference in the fluorescent label compounds as compared to the conventional method shown in (iv) of Table 3 (FIG. 2).

[0104] Also, as shown in Table 3, it was found that the accuracy becomes higher when the concentration ratio of non-labeled substrate/labeled substrate (non-labeled substrate/labeled substrate=5/1) was high as compared to that when the concentration ratios of the non-labeled substrate/labeled substrate for Cy3 and Cy5 were set at the same level, and the concentration ratio of the non-labeled substrate/labeled substrate was set low (non-labeled substrate/labeled substrate=2/1). Moreover, it was found that the accuracy was further increased when the concentration ratios of the non-labeled substrate/labeled substrate were set at different levels for Cy3 and Cy5, and when the concentration ratio of the non-labeled substrate/labeled substrate for Cy3 was 2/1 and the concentration ratio of the non-labeled substrate/labeled substrate for Cy5 was 5/1.

[0105] (2) Comparison with Commercially Available Kit

[0106] A Cy3-labeled cDNA probe and a Cy5-labeled cDNA probe were prepared with the mRNA of Example 1 as a template using Cyscribe First-strand cDNA Labeling Kit (manufactured by Amersham-Pharmacia), a commercially available kit for preparing CyDye-labeled cDNA probes, in accordance with the instruction manual attached to the kit. The hybridization with the DNA microarray, washing, scanning and analysis were carried out under the same conditions as those described in Example 1. The results of Scatter Plot are shown in FIG. 3. FIG. 3 is a diagram showing signal intensities obtained when each fluorescent label was used. In the figure, the X-axis represents a Cy3 signal intensity, and the Y-axis represents a Cy5 signal intensity. Also, an open circle (◯) represents a spot exhibiting a significant signal intensity, a cross (+) represents a spot exhibiting a non-significant signal intensity, a solid line is a line showing an expression ratio of 1:1, a broken line is a line showing an expression ratio of 2:1 or 1:2, and a dotted line is a line showing an expression ratio of 3:1 or 1:3.

[0107] As in the case of the above (1), the expression ratio has to be 1:1 for all of the genes. However, as shown in FIG. 3, 57% of the genes showed a difference in expression of 2 folds or more when the Cy3-labeled cDNA probe was used and when the Cy5-labeled cDNA probe was used, and the Cy3/Cy5 ratio which was deduced at a 99% convergence in distribution of significant spots was shown to have a value of 8.5.

[0108] It is found, however, that the variance of spots is notably smaller in FIG. 1 as compared to that of FIG. 3. In other words, it was found that the labeling method shown in (ii) of Table 3 (FIG. 1) is a method showing smaller difference in labeling efficiency caused by the difference in the fluorescent label compounds, as compared to that of the labeling method using the commercially available kit (FIG. 3).

EXAMPLE 3

[0109] In order to confirm that the results obtained in Example 1 are phenomena generally found for any sorts of DNA chips without being altered by the properties (degree of background and the like) of particular substrates of DNA chips and DNA microarrays and the binding manner of the DNA with the substrate, the studies were conducted in the same manner as in Example 1 using two kinds of DNA chips having different substrates.

[0110] The preparation of the DNA chips was carried out as follows. Concretely, a slide glass into which an activated carboxyl group was introduced was prepared in accordance with the method described in WO 01/02538. Next, approximately 770 kinds of human cancer-related genes listed in Tables 4 to 60 were selected, and primer pairs were designed so that approximately 300 bp regions shown in Tables 4 to 60 can be amplified on the basis of the nucleotide sequences of these genes.

[0111] Tables 4 to 60 are tables showing the names of genes and accession numbers (GenBank Accession Numbers) of the cancer-related genes and the specific gene regions selected for each of the genes, wherein the regions are shown by the corresponding numbers of nucleic acids registered in the database.

TABLE 4
	Accession	Selected Gene
Name of Gene	Number	Region
SFRS protein kinase 2	NM_003138	2251-2550
matrix metalloproteinase	NM_004994	1893-2192
9(gelatinase B, 92kD gelatinase,
92kD type IV collagenase)
fibroblast growth factor 2	NM_002006	5759-6058
(basic)
mitogen-activated protein	NM_002753	2028-2327
kinase 10
hexabrachion (tenascin C,	NM_002160	6557-6856
cytotactin)
MAD (mothers against deca-	NM_005359	1697-1996
pentaplegic, Drosophila)
homolog 4
Human clone 23878 mRNA	U79251	696-397
sequence
tumor protein p53 (Li-Fraumeni	NM_000546	1602-1901
syndrome)
catenin (cadherin-associated	NM_004389	2710-3009
protein), alpha 2
cell adhesion molecule with	NM_006614	6316-6615
homology to L1CAM (close
homologue of L1)
contactin 2 (axonal)	NM_005076	3922-4221
protein phosphatase 2, regu-	NM_006243	2150-2449
latory subunit B (B56), alpha
isoform
v-myc avian myelocytomatosis	Y00664	1066-1365
viral related oncogene,
neuroblastoma derived
mitogen-activated protein	NM_002748	2470-2769
kinase 6
serine (or cysteine) pro-	NM_002575	1307-1606
teinase inhibitor, clade B
(ovalbumin), member 2
v-yes-1 Yamaguchi sarcoma	NM_005433	4214-4513
viral oncogene homolog 1

[0112]

TABLE 5
	Accession	Selected
Name of Gene	Number	Gene Region
caspase 3, apoptosis-related	NM_004346	1984-2283
cysteine protease
matrix metalloproteinase 17	NM_016155	1342-1641
(membrane-inserted)
cadherin 13, H-cadherin (heart)	NM_001257	3614-3913
eukaryotic translation elongation	NM_001961	1843-2142
factor 2
eukaryotic translation elongation	NM_001961	1842-2141
factor 2
baculoviral IAP repeat-containing 2	NM_001166	3022-3321
diphtheria toxin receptor (heparin-binding	NM_001945	1037-1336
epidermal growth factor-like growth factor)
mitogen-activating protein kinase	NM_004579	1509-1808
kinase kinase kinase 2
phosphoinositide-3-kinase,	M61906	1838-1539
regulatory subunit,
polypeptide 1 (p85 alpha)
IMP (inosine monophosphate)	NM_000883	2039-2338
dehydrogenase 1
ESTs, Highly similar to CAD8_HUMAN	AA552988	522-223
CADHERIN-8 PRECURSOR [H. sapiens]
lunatic fringe (Drosophila) homolog	U94354	1-300
lunatic fringe (Drosophila) homolog	U94354	13-312
bone morphogenetic protein 1	NM_006129	2373-2672
bone morphogenetic protein 1	NM_006129	2374-2673
interleukin 13 receptor, alpha 2	NM_000640	836-1135
hepatocyte growth factor(hepapoietin A;	X16323	1555-1854
scatter factor)
5′ nucleotidase (CD73)	NM_002526	3153-3452

[0113]

TABLE 6
	Accession	Selected Gene
Name of Gene	Number	Region
TRK-fused gene	NM_006070	999-1298
vascular cell adhesion molecule 1	NM_001078	2271-2570
vascular endothelial growth factor C	NM_005429	870-1169
G protein-coupled receptor 19	NM_006143	1065-1364
leucyl/cystinyl aminopeptidase	NM_005575	2164-2463
Human insulin-like growth factor	L27560	3080-2781
binding protein 5 (IGFBP5) mRNA
mitogen-activated protein kinase	U39657	1283-1582
kinase 6
choline kinase	NM_001277	570-869
Human clone 23734 mRNA sequence	U79292	1385-1086
p53-induced protein	NM_006034	1885-2184
E74-like factor 4(ets domain	NM_001421	3231-3530
transcription factor)
vascular cell adhesion molecule 1	NM_001078	2271-2570
AXL receptor tyrosine kinase	NM_021913	2168-2467
SKI-INTERACTING PROTEIN	NM_012245	1186-1485
peroxiredoxin 3	NM_006793	1005-1304
cell division cycle 34	L22005	528-827
general transcription factor IIB	NM_001514	333-632
baculoviral IAP repeat-containing 1	NM_004536	4933-5232
excision repair cross-complementing	NM_000122	1915-2214
rodent repair deficiency, complementation
group 3 (xeroderma pigmentosum group B
complementing)
manic fringe (Drosophila) homolog	NM_002405	1003-1302

[0114]

TABLE 7
	Accession	Selected Gene
Name of Gene	Number	Region
platelet-derived growth factor	NM_006206	5508-5807
receptor, alpha polypeptide
endothelin 3	NM_000114	1586-1885
plasminogen-like	NM_002665	225-524
platelet-derived growth factor	NM_002608	2898-3197
beta polypeptide (simian sarcoma
viral (v-sis) oncogene homolog)
platelet-derived growth factor	NM_002608	2897-3196
beta polypeptide (simian sarcoma
viral (v-sis) oncogene homolog)
cyclin E1	M74093	1623-1324
natural killer cell group	NM_005601	67-366
7 sequence
caspase 7, apoptosis-related	NM_001227	1203-1502
cysteine protease
nerve growth factor, beta	NM_002506	239-538
polypeptide
nerve growth factor, beta	NM_002506	238-537
polypeptide
exportin 1 (CRM1, yeast, homolog)	NM_003400	3542-3841
EphB4	NM_004444	3628-3927
TNF receptor-associated factor 2	NM_021138	1749-2048
small inducible cytokine subfamily	NM_004590	1196-1495
A (Cys—Cys), member 16
betaine-homocysteine	NM_001713	1353-1652
methyltransferase
FBJ murine osteosarcoma viral	NM_006732	3447-3746
oncogene homolog B
small inducible cytokine subfamily	NM_005408	470-769
A (Cys—Cys), member 13

[0115]

TABLE 8
	Accession	Selected Gene
Name of Gene	Number	Region
Rho-associated, coiled-coil	NM_005406	1646-1945
containing protein kinase 1
GTP-binding protein homologous	NM_006822	1328-1627
to Saccharomyces cerevisiae SEC4
lysophospholipase-like	NM_007283	802-1101
P450 (cytochrome) oxidoreductase	AF258341	1733-2032
glutathione S-transferase A2	AL109918	1472-1771
deoxyribonuclease I-like 3	NM_004944	306-605
growth arrest and DNA-damage-	NM_006705	419-718
inducible, gamma
growth arrest and DNA-damage-	NM_006705	418-717
inducible, gamma
plasminogen	NM_000301	2380-2679
cell division cycle 27	NM_001256	193-492
caveolin 2	NM_001233	533-832
serine/threonine kinase 4	NM_006282	835-1134
caspase 1, apoptosis-related	NM_001223	642-941
cysteine protease (interleukin
1, beta, convertase)
excision repair cross-complementing	NM_001983	369-668
rodent repair deficiency, complementation
group 1 (includes overlapping antisense
sequence)
signal sequence receptor, alpha	NM_003144	368-667
(translocon-associated protein alpha)
interferon-related developmental	NM_001550	1091-1390
regulator 1
casein kinase 2, alpha 1 polypeptide	NM_001895	40-339

[0116]

TABLE 9
	Accession	Selected Gene
Name of Gene	Number	Region
collagen, type III, alpha 1	NM_000090	4033-4332
(Ehlers-Danlos syndrome type
IV, autosomal dominant)
jun B proto-oncogene	NM_002229	1229-1528
wingless-type MMTV integration	NM_003391	1574-1873
site family member 2
putative chemokine receptor;	NM_006018	249-548
GTP-binding protein
dihydrofolate reductase	NM_000791	455-754
translocated promoter region	NM_003292	331-630
(to activated MET oncogene)
hyaluronan-mediated motility	NM_012484	1545-1844
receptor (RHAMM)
intercellular adhesion	NM_000873	32-331
molecule 2
peripheral myelin protein 22	NM_000304	1011-1310
protein kinase, DNA-activated,	U47077	13130-13429
catalytic polypeptide
glutathione S-transferase pi	NM_000852	153-452
transforming growth factor,	NM_000660	1404-1703
beta 1
Glutamate receptor interacting	AJ133439	2231-2530
protein
programmed cell death 10	NM_007217	711-1010
mouse double minute 2, human	NM_002392	128-427
homolog of; p53-binding protein
mouse double minute 2, human	NM_002392	129-428
homolog of; p53-binding protein
arachidonate 5-lipoxygenase	NM_000698	1235-1534

[0117]

TABLE 10
	Accession	Selected Gene
Name of Gene	Number	Region
arachidonate 5-lipoxygenase	NM_000698	1234-1533
insulin induced gene 1	NM_005542	1393-1692
insulin induced gene 1	NM_005542	1392-1691
guanine monphosphate synthetase	NM_003875	1913-2212
chromodomain helicase DNA	NM_001270	5018-5317
binding protein 1
glutathione S-transferase	NM_000853	276-575
theta 1
collagen, type VI, alpha 3	NM_004369	9819-10118
fibronectin 1	X02761	6690-6989
jagged 1 (Alagille syndrome)	NM_000214	5239-5538
KIAA0128 protein; septin 2	D50918	1678-1977
deoxyribonuclease I-like 1	NM_006730	2058-2357
protocadherin 1 (cadherin-like 1)	NM_002587	3764-4063
epidermal growth factor	NM_004447	3270-3569
receptor pathway substrate 8
wingless-type MMTV integration	NM_003391	1574-1873
site family member 2
selectin L (lymphocyte adhesion	NM_000655	1451-1750
molecule 1)
TEK tyrosine kinase, endothelial	NM_000459	3599-3898
(venous malformations, multiple
cutaneous and mucosal)
TEK tyrosine kinase, endothelial	NM_000459	3598-3897
(venous malformations, multiple
cutaneous and mucosal)
protease, serine, 22	NM_006025	2021-2320
leukemia inhibitory factor	NM_002309	2967-3266
(cholinergic differentiation
factor)

[0118]

TABLE 11
	Accession	Selected Gene
Name of Gene	Number	Region
leukemia inhibitory factor	NM_002309	2966-3265
(cholinergic differentiation
factor)
SH3-domain binding protein 2	AB000462	4144-4443
integrin, alpha L(antigen CD11A	NM_002209	4320-4619
(p180), lymphocyte function-
associated antigen 1; alpha
polypeptide)
collagen, type XVI, alpha 1	NM_001856	113-412
nerve growth factor receptor	NM_002507	2719-3018
(TNFR superfamily, member 16)
neuregulin 1	NM_013957	857-1156
GTP-binding protein homologous	NM_006822	468-767
to Saccharomyces cerevisiae SEC4
ras homolog gene family, member G	NM_001665	905-1204
(rho G)
laminin, gamma 1 (formerly LAMB2)	NM_002293	833-1132
actin related protein 2/3 complex,	NM_005731	640-939
subunit 2 (34 kD)
peripheral myelin protein 22	NM_000304	1427-1726
transcription elongation factor A	NM_006756	1748-2047
(SII), 1
integrin, alpha 8	L36531	781-1080
adrenergic, beta, receptor kinase 1	NM_001619	1901-2200
adrenergic, beta, receptor kinase 1	NM_001619	1900-2199
protocadherin gamma subfamily C, 3	NM_002588	1731-2030
glia maturation factor, beta	NM_004124	3448-3747
mitogen-activated protein kinase 7	NM_002749	2161-2460
mitogen-activated protein kinase 7	NM_002749	2160-2459

[0119]

TABLE 12
	Accession	Selected Gene
Name of Gene	Number	Region
purine-rich element binding	NM_005859	618-917
protein A
purine-rich element binding	NM_005859	619-918
protein A
catenin (cadherin-associated	NM_004389	3410-3709
protein), alpha 2
tumor protein p53-binding	NM_005657	1617-1916
protein, 1
activin A receptor type	NM_000020	348-647
II-like 1
caspase 4, apoptosis-related	AL050391	3835-4134
cysteine protease
keratin 14 (epidermolysis bullosa	NM_000526	803-1102
simplex, Dowling-Meara, Koebner)
myeloid differentiation primary	U70451	1851-2150
response gene(88)
thrombospondin 2	NM_003247	4969-5268
breast cancer 2, early onset	NM_000059	10426-10725
G protein-coupled receptor	NM_006564	1471-1770
topoisomerase (DNA) II binding	NM_007027	4901-5200
protein
baculoviral IAP repeat-containing 3	AF070674	4618-4917
non-metastatic cells 4, protein	NM_005009	570-869
expressed in
tumor necrosis factor(ligand)	NM_003810	371-670
superfamily, member10
CDC6(cell division cycle 6, S. cerevisiae)	NM_001254	982-1281
homolog
a disintegrin and metalloproteinase	NM_003816	2763-3062
domain 9 (meltrin gamma)
CD59 antigen p18-20(antigen identified	NM_000611	803-1102
by mono-clonal antibodies 16.3A5, EJ16,
EJ30, EL32 and G344)

[0120]

TABLE 13
	Accession	Selected Gene
Name of Gene	Number	Region
glycophorin B (includes Ss blood group)	NM_002100	88-387
dual-specificity tyrosine-(Y)-	NM_003582	1742-2041
phosphorylation regulated kinase 3
transforming growth factor, beta	NM_003243	2704-3003
receptor III (betaglycan, 300 kD)
transforming growth factor, beta	NM_003243	2703-3002
receptor III (betaglycan, 300 kD)
integrin, alpha 1	X68742	3079-2780
tumor protein p53-binding protein, 2	NM_005426	4147-4446
mannosidase, alpha, class 2A,	NM_002372	3502-3801
member 1
Rho GTPase activating protein 4	NM_001666	2348-2647
Rho GTPase activating protein 4	NM_001666	2347-2646
Homo sapiens cDNA: FLJ21562 fis,	AK025215	1577-1876
clone COL06420
kinase suppressor of ras	U43586	748-1047
guanine nucleotide binding protein	NM_002067	1228-1527
(G protein), alpha 11 (Gq class)
GTP-binding protein	NM_012341	1528-1827
B-cell CLL/lymphoma 2	NM_000633	2342-2641
insulin-like growth factor binding	NM_000597	670-969
protein2(36 kD)
GATA-binding protein 6	NM_005257	2138-2437
BCL2-antagonist/killer 1	NM_001188	1616-1915
phospholipase A2, group VII(platelet-	NM_005084	1036-1335
activating factor acetylhydrolase, plasma)

[0121]

TABLE 14
	Accession	Selected Gene
Name of Gene	Number	Region
desmoplakin (DPI, DPII)	NM_004415	8024-8323
breast cancer 1, early onset	NM_007295	7045-7344
caspase 10, apoptosis-related	NM_001230	2967-3266
cysteine protease
EphA2	NM_004431	2664-2963
EphA2	NM_004431	2663-2962
tyrosine 3-monooxygenase/tryptophan	NM_003406	2063-2362
5-monooxygenase activation protein,
zeta polypeptide
inhibitor of growth 1 family, member 1	NM_005537	840-1139
HSPC070 protein	NM_014160	2016-2315
RAD52 (S. cerevisiae) homolog	NM_002879	2293-2592
insulin-like growth factor 2	X07868	819-1118
(somatomedin A)
HMT1(hnRNP methyltransferase,	NM_001536	595-894
S. cerevisiae )-like 2
CHK1 (checkpoint, S. pombe) homolog	NM_001274	1271-1570
CGI-150 protein	NM_016080	1883-2182
vitronectin (serum spreading factor,	NM_000638	931-1230
somatomedin B, complement S-protein)
vitronectin(serum spreading factor,	NM_000638	932-1231
somatomedin B, complement S-protein)
cyclin D2	NM_001759	706-1005
dual specificity phosphatase 8	NM_004420	2065-2364
cadherin 1, type 1, E-cadherin	NM_004360	4479-4778
(epithelial)

[0122]

TABLE 15
	Accession	Selected Gene
Name of Gene	Number	Region
cadherin 11, type 2, OB-cadherin	D21255	3033-3332
(osteoblast)
collagen, type XVII, alpha 1	AL138761	4727-5026
collagen, type XI, alpha 2	AL031228	6121-6420
retinoblastoma-binding protein 6	NM_006910	114-413
guanine nucleotide binding	NM_005275	1411-1710
protein-like 1
chorionic gonadotropin, beta	NM_000737	152-451
polypeptide
chorionic gonadotropin, beta	NM_000737	151-450
polypeptide
interferon regulatory factor 5	NM_002200	186-485
Human DNA sequence from clone	AL157902	2720-3019
RP4-675C20 on chromosome
1p13.2. Contains the 3′ end of
the MAN1A2 gene for mannosidase
alpha 1A2, a pseudogene similar
to predicted fly, worm and yeast
genes, ESTs, STSs and GSSs
cathepsin D (lysosomal aspartyl	NM_001909	356-655
protease)
bone morphogenetic protein re-	NM_001204	2722-3021
ceptor, type II (serine/
threonine kinase)
v-kit Hardy-Zuckerman 4 feline	NM_000222	4386-4685
sarcoma viral oncogene homolog
3-phosphoinositide dependent	NM_002613	1377-1676
protein kinase-1
FYN oncogene related to SRC,	Z97989	1706-2005
FGR, YES
v-kit Hardy-Zuckerman 4 feline	NM_000222	4386-4685
sarcoma viral oncogene homolog
matrix metalloproteinase 14	NM_004995	2435-2734
(membrane-inserted)

[0123]

TABLE 16
	Accession	Selected Gene
Name of Gene	Number	Region
CDC7 (cell division cycle 7,	NM_003503	2870-3169
S. cerevisiae , homolog)-like 1
allograft inflammatory factor 1	NM_004847	850-1149
mitogen-activated protein	NM_002757	1436-1735
kinase kinase 5
cell division cycle 2-like 5	AJ297709	1737-2036
(cholinesterase-related cell
division controller)
FYN oncogene related to SRC,	Z97989	2088-2387
FGR, YES
Ser-Thr protein kinase related	NM_003607	2444-2743
to the myotonic dystrophy pro-
tein kinase
tyrosinase (oculocutaneous	NM_000372	1665-1964
albinism IA)
v-yes-1 Yamaguchi sarcoma	NM_005433	4215-4514
viral oncogene homolog1
multifunctional polypeptide	NM_006452	492-791
similar to SAICAR synthetase
and AIR carboxylase
glucuronidase, beta	NM_000181	247-546
glucuronidase, beta	NM_000181	248-547
FYN oncogene related to SRC,	Z97989	1706-2005
FGR, YES
hypoxanthine phosphoribosyl-	NM_000194	531-830
transferase 1 (Lesch-Nyhan
syndrome)
inhibitor of growth 1 family,	NM_005537	1408-1707
member 1
sema domain, immunoglobulin	NM_006378	3258-3557
domain (Ig), transmembrane
domain (TM) and short cyto-
plasmic domain, (semaphorin)
4 D
RAB36, member RAS oncogene	NM_004914	1300-1599
family

[0124]

TABLE 17
	Accession	Selected Gene
Name of Gene	Number	Region
H2A histone family, member L	NM_003512	550-849
matrix metalloproteinase 13	NM_002427	2193-2492
(collagenase 3)
interferon (alpha, beta and	NM_000629	2454-2753
omega) receptor 1
neutral sphingomyelinase	NM_003580	2466-2765
(N-SMase) activation asso-
ciated factor
interleukin 15	NM_000585	617-916
interleukin 15	NM_000585	618-917
cyclin-dependent kinase	NM_001262	1211-1510
inhibitor 2C (p18, inhibits
CDK4)
silver (mouse homolog) like	NM_006928	1427-1726
dishevelled 3 (homologous to	NM_004423	3247-3546
Drosophila dsh)
death-associated protein 6	NM_001350	1939-2238
excision repair cross-comple-	NM_000123	2973-3272
menting rodent repair deficiency,
complementation group 5 (xeroderma
pigmentosum, complementation
group G (Cockayne syndrome))
ankyrin 1, erythrocytic	NM_000037	7923-8222
lysozyme (renal amyloidosis)	NM_000239	1188-1487
Homo sapiens cDNA FLJ11848	AK021910	1129-1428
fis, clone HEMBA 1006708,
weakly similar to HYPO-
THETICAL 46.4 KD TRP-ASP
REPEATS CONTAINING PROTEIN
IN PMC1-TFG2 INTERGENIC
REGION
GTP-binding protein ragB	NM_016656	172-471

[0125]

TABLE 18
	Accession	Selected Gene
Name of Gene	Number	Region
transcription factor Dp-1	NM_007111	40-339
insulin-like growth factor	X07868	101-400
2 (somatomedin A)
glycophorin A (includes MN	NM_002099	399-698
blood group)
small inducible cytokine	NM_005624	64-363
subfamily A (Cys-Cys),
member 25
metastasis associated 1	NM_004689	1400-1699
metastasis associated 1	NM_004689	1399-1698
adaptor-related protein	NM_001128	3264-3563
complex 1, gamma1 subunit
far upstream element (FUSE)	U69127	2280-2579
binding protein 3
collagen, type XVIII, alpha 1	AF018081	4732-5031
collagen, type XVIII, alpha 1	AF018081	4731-5030
protein phosphatase 3 (formerly	NM_005605	282-581
2B), catalytic subunit, gamma
isoform (calcineurin A gamma)
wingless-type MMTV integration	NM_003391	1971-2270
site family member 2
ras homolog gene family, member H	NM_004310	221-520
E2F transcription factor 3	NM_001949	4003-4302
v-Ki-ras2 Kirsten rat sarcoma 2	NM_004985	674-973
viral oncogene homolog
replication factor C (activator 1)	NM_002916	1090-1389
4 (37 kD)
CASP8 and FADD-like apoptosis	Y14039	557-856
regulator

[0126]

TABLE 19
	Accession	Selected Gene
Name of Gene	Number	Region
jun B proto-oncogene	NM_002229	1229-1528
jun B proto-oncogene	NM_002229	1228-1527
active BCR-related gene	NM_021962	4670-4969
active BCR-related gene	NM_021962	4669-4968
retinoic acid- and interferon-	NM_012420	2990-3289
inducible protein (58 kD)
CGI-39 protein; cell death-	NM_015965	217-516
regulatory protein GRIM19
chromogranin B (secretogranin 1)	NM_001819	1958-2257
phosphatase and tensin homolog	NM_000314	2666-2965
(mutated in multiple advanced
cancers 1)
fibroblast growth factor 2 (basic)	NM_002006	3424-3723
matrix metalloproteinase 3	NM_002422	860-1159
(stromelysin 1, progelatinase)
interleukin 1, beta	NM_000576	1048-1347
Notch (Drosophila) homolog 3	NM_000435	3583-3882
Notch (Drosophila) homolog 3	NM_000435	3582-3881
epidermal growth factor receptor	NM_005228	5063-5362
(avian erythro-blastic leukemia
viral (v-erb-b) oncogene homolog)
wingless-type MMTV integration	NM_003392	1965-2264
site family, member 5A
cadherin 2, type 1, N-cadherin	S42303	3171-3470
(neuronal)

[0127]

TABLE 20
	Accession	Selected Gene
Name of Gene	Number	Region
cadherin 2, type 1, N-cadherin	S42303	3170-3469
(neuronal)
keratin 13	NM_002274	1286-1585
elastase 3B	NM_007352	184-483
CD44 antigen (homing function	AJ251595	2468-2767
and Indian blood group system)
mitogen-activated protein kinase	NM_005204	55-354
kinase kinase 8
mitogen-activated protein kinase	NM_005204	54-353
kinase kinase 8
integrin, beta 8	NM_002214	2753-3052
keratin 13	NM_002274	1292-1591
RAB2, member RAS oncogene family	NM_002865	590-889
RAB2, member RAS oncogene family	NM_002865	589-888
B-cell CLL/lymphoma 2	NM_000633	5307-5606
mitogen-activated protein kinase	NM_003954	3516-3815
kinase kinase14
platelet-derived growth factor	NM_002608	2898-3197
beta polypeptide (simian sarcoma
viral (v-sis) oncogene homolog)
platelet-derived growth factor	NM_002608	2897-3196
beta polypeptide (simian sarcoma
viral (v-sis) oncogene homolog)
uridine monophosphate kinase	NM_012474	165-464
microsomal glutathione S-transferase 2	NM_002413	223-522
Fas-activated serine/threonine kinase	NM_006712	1098-1397
Fas-activated serine/threonine kinase	NM_006712	1099-1398
EphB2	AF025304	3508-3807

[0128]

TABLE 21
	Accession	Selected Gene
Name of Gene	Number	Region
lymphotoxin alpha (TNF	NM_000595	845-1144
superfamily, member 1)
lymphotoxin alpha (TNF	NM_000595	846-1145
superfamily, member 1)
general transcription factor	NM_001515	764-1063
IIH, polypeptide 2
(44 kD subunit)
cathepsin L	NM_001912	1063-1362
v-akt murine thymoma viral	NM_005163	562-861
oncogene homolog 1
guanylate binding protein	NM_004120	696-995
2, interferon-inducible
guanylate binding protein	NM_004120	697-996
2, interferon-inducible
angiogenin, ribonuclease,	NM_001145	95-394
RNase A family, 5
eukaryotic translation	NM_003908	857-1156
initiation factor 2,
subunit 2 (beta, 38 kD)
retinoblastoma 1 (including	NM_000321	3892-4191
osteosarcoma)
mitogen-activated protein	AL157438	1097-798
kinase 1
tumor necrosis factor,	NM_007115	447-746
alpha-induced protein 6
neuronal Shc	NM_016848	1106-1405
heterogeneous nuclear	NM_005520	1459-1758
ribonucleoprotein H1 (H)
v-jun avian sarcoma virus	NM_002228	2823-3122
17 oncogene homolog
transmembrane trafficking	NM_006827	125-424
protein
BCL2/adenovirus E1B 19 kD-	NM_004052	468-767
interacting protein 3
ataxia telangiectasia mutated	U82828	12746-13045
(includes complementation
groups A, C and D)
protein kinase, cAMP-dependent,	NM_002731	2330-2629
catalytic, beta
IKK-related kinase epsilon;	NM_014002	2920-3219
inducible IkappaB
kinase

[0129]

TABLE 22
	Accession	Selected Gene
Name of Gene	Number	Region
fibroblast growth factor 7	NM_002009	1451-1750
(keratinocyte growth factor)
primase, polypeptide 1 (49 kD)	NM_000946	812-1111
envoplakin	NM_001988	6100-6399
envoplakin	NM_001988	6099-6398
Human DNA sequence from clone	AL157414	2443-2742
RP11-560A15 on chromosome 20
Contains part of a novel gene,
the 3′ part of the BMP7 (bone
morphogenetic protein 7
(osteogenic protein 1)) gene,
ESTs, STSs, GSSs and a CpG island
topoisomerase (DNA) II	NM_001067	3593-3892
alpha (170 kD)
wee1 + (S. pombe) homolog	X62048	2495-2794
caveolin 1, caveolae protein,	NM_001753	460-759
22 kD
integrin, alpha 7	NM_002206	3585-3884
integrin, alpha 7	NM_002206	3584-3883
reticulon 3	NM_006054	1903-2202
30 kDa protein	NM_018447	378-677
neutral sphingomyelinase (N-SMase)	NM_003580	993-1292
activation associated factor
cell division cycle 25C	NM_001790	1288-1587
dishevelled 3 (homologous to	NM_004423	4927-5226
Drosophila dsh)

[0130]

TABLE 23
	Accession	Selected Gene
Name of Gene	Number	Region
ras homolog gene family,	NM_005168	42-341
member E
serine(or cysteine)proteinase	NM_000624	344-643
inhibitor, clade A (alpha-1
antiproteinase, antitrypsin),
member 5
cell growth regulatory with	NM_006568	503-802
ring finger domain
pro-platelet basic protein (includes	NM_002704	252-551
platelet basic protein, beta-
thromboglobulin, connective
tissue-activating peptide III,
neutrophil-activating peptide-2)
microtubule-associated protein,	NM_012325	1469-1768
RP/EB family, member 1
baculoviral IAP repeat-containing 3	AF070674	2981-3280
caspase 3, apoptosis-related cysteine	NM_004346	647-946
protease
H2B histone family, member Q	NM_003528	447-746
centromere protein F	NM_005196	9796-10095
(350/400 kD, mitosin)
platelet-derived growth factor	NM_002607	1742-2041
alpha polypeptide
H. sapiens mRNA for hcgVIII protein	X92110	515-216
delta-like homolog (Drosophila)	NM_003836	726-1025
delta-like homolog (Drosophila)	NM_003836	727-1026
tumor necrosis factor(TNF	NM_000594	1122-1421
superfamily, member 2)
baculoviral IAP repeat-containing 2	NM_001166	1489-1788
retinoid X receptor, alpha	NM_002957	1197-1496
cell growth regulatory with ring	NM_006568	503-802
finger domain

[0131]

TABLE 24
	Accession	Selected Gene
Name of Gene	Number	Region
kinase insert domain receptor	NM_002253	4800-5099
(a type III receptor tyrosine kinase)
KIAA0001 gene product; putative	NM_014879	1670-1969
G-protein-coupled receptor; G protein
coupled receptor for UDP-glucose
ligase III, DNA, ATP-dependent	NM_013975	2981-3280
ligase III, DNA, ATP-dependent	NM_013975	2982-3281
matrix metalloproteinase 7	Z11887	365-664
(matrilysin, uterine)
RAB32, member RAS oncogene family	NM_006834	455-754
transforming growth factor, beta 3	NM_003239	1970-2269
sema domain, immunoglobulin domain	NM_006379	3212-3511
(Ig), short basic domain, secreted,
(semaphorin) 3C
integrin beta 3 binding protein	NM_014288	580-879
(beta3-endonexin)
tissue inhibitor of metalloproteinase	NM_000362	2599-2898
3 (Sorsby fundus dystrophy,
pseudoinflammatory)
chondroitin sulfate proteoglycan 2	U16306	10927-11226
(versican)
CD58 antigen, (lymphocyte function-	NM_001779	229-528
associated antigen 3)
glycoprotein A repetitions predominant	NM_005512	3656-3955
glycoprotein A repetitions predominant	NM_005512	3655-3954
cadherin 11, type 2, OB-cadherin	D21255	2919-3218
(osteoblast)
STAT induced STAT inhibitor-2	NM_003877	509-808

[0132]

TABLE 25
	Accession	Selected Gene
Name of Gene	Number	Region
collagen, type IV, alpha 1	NM_001845	2609-2908
interleukin 1, beta	NM_000576	218-517
collagen, type XVII, alpha 1	AL138761	5168-5467
laminin, beta 2 (laminin S)	NM_002292	5273-5572
histone deacetylase 2	NM_001527	1556-1855
retinoblastoma-binding protein 1	NM_002892	4077-4376
Wilms tumor 1	X51630	1849-2148
monokine induced by gamma interferon	NM_002416	2054-2353
hemopoietic cell kinase	NM_002110	1499-1798
hemopoietic cell kinase	NM_002110	1498-1797
syndecan 4 (amphiglycan, ryudocan)	NM_002999	2316-2615
mitogen-activated protein kinase	NM_005923	4005-4304
kinase kinase 5
carcinoembryonic antigen-related	M18216	1941-2240
cell adhesion molecule 6(non-
specific cross reacting antigen)
signal sequence receptor, alpha	NM_003144	2229-2528
(translocon-associated protein
alpha)
ectodermal-neural cortex	NM_003633	1680-1979
(with BTB-like domain)
ectodermal-neural cortex	NM_003633	1681-1980
(with BTB-like domain)
protein phosphatase 2, regulatory	NM_006244	2006-2305
subunit B (B56), beta isoform
peroxisome proliferative activated	NM_005037	744-1043
receptor, gamma
cadherin 17, LI cadherin	NM_004063	2283-2582
(liver-intestine)
syndecan 1	NM_002997	1547-1846

[0133]

TABLE 26
	Accession	Selected Gene
Name of Gene	Number	Region
c-src tyrosine kinase	NM_004383	2049-2348
guanine nucleotide binding	NM_002070	1453-1752
protein (G protein), alpha
inhibiting activity polypeptide 2
protein kinase, AMP-activated,	NM_002733	697-996
gamma 1 non-catalytic subunit
interleukin 8	NM_000584	760-1059
phosphatidylinositol glycan,	NM_002643	509-808
class F
phosphatidylinositol glycan,	NM_002643	508-807
class F
protein kinase C-like 2	NM_006256	2606-2905
tousled-like kinase 1	NM_012290	3183-3482
TNF receptor-associated factor 5	NM_004619	3668-3967
growth factor receptor-bound	D86962	4997-5296
protein 10
cytokeratin 20	X73502	871-1170
placental growth factor, vascular	NM_002632	1095-1394
endothelial growth factor-
related protein
placental growth factor, vascular	NM_002632	1096-1395
endothelial growth factor-
related protein
matrix metalloproteinase 1	NM_002421	711-1010
(interstitial collagenase)
Tat-interacting protein (30 kD)	NM_006410	424-723
adenylate cyclase 6	NM_015270	6138-6437
integrin-linked kinase	NM_004517	952-1251
keratin 5 (epidermolysis bullosa	NM_000424	2150-2449
simplex, Dowling-Meara/Kobner/
Weber-Cockayne types)

[0134]

TABLE 27
	Accession	Selected Gene
Name of Gene	Number	Region
patched (Drosophila) homolog	U43148	4363-4662
IGF-II mRNA-binding protein 3	NM_006547	2860-3159
TRAF interacting protein	NM_005879	1359-1658
TRAF interacting protein	NM_005879	1358-1657
mutS (E. coli) homolog 2 (colon cancer,	NM_000251	2307-2606
nonpolyposis type 1)
mutS (E. coli) homolog 2 (colon cancer,	NM_000251	2306-2605
nonpolyposis type 1)
CD27-binding (Siva) protein	NM_006427	69-368
CD27-binding (Siva) protein	NM_006427	68-367
eukaryotic translation initiation factor 4E	NM_001968	355-654
topoisomerase (DNA) 1	NM_003286	3019-3318
brain-derived neurotrophic factor	NM_001709	882-1181
heat shock transcription factor 2	NM_004506	1646-1945
Gardner-Rasheed feline sarcoma viral	NM_005248	1911-2210
(v-fgr) oncogene homolog
fragile histidine triad gene	NM_002012	257-556
multiple endocrine neoplasia I	NM_000244	2411-2710
v-rel avian reticuloendotheliosis viral	NM_006509	1216-1515
oncogene homolog B (nuclear factor of
kappa light polypeptide gene enhancer
in B-cells 3)
osteoclast stimulating factor 1	NM_012383	28-327

[0135]

TABLE 27
	Accession	Selected Gene
Name of Gene	Number	Region
glutaredoxin (thioltransferase)	NM_002064	1-300
glutaredoxin (thioltransferase)	NM_002064	2-301
xeroderma pigmentosum,	NM_004628	3067-3366
complementation group C
myxovirus (influenza) resistance	NM_002463	2096-2395
2, homolog of murine
nuclear protein, ataxia-	NM_002519	4610-4909
telangiectasia locus
RAP1A, member of RAS oncogene	NM_002884	1023-1322
family
suppression of tumorigenicity	NM_006850	1177-1476
16 (melanoma differentiation)
signal transducer and activator	NM_012448	2327-2626
of transcription 5B
MAP-kinase activating death domain	NM_003682	5354-5653
CDC-like kinase 2	NM_003993	1334-1633
CDC-like kinase 2	NM_003993	1333-1632
CD44 antigen (homing function	AJ251595	1772-2071
and Indian blood group system)
PCTAIRE protein kinase 1	NM_006201	1263-1562
PCTAIRE protein kinase 1	NM_006201	1262-1561
plasminogen activator, urokinase	NM_002658	1929-2228
tumor necrosis factor receptor	NM_000043	2212-2511
superfamily, member 6
interferon-stimulated transcription	NM_006084	1061-1360
factor 3, gamma (48 kD)

[0136]

TABLE 29
	Accession	Selected Gene
Name of Gene	Number	Region
chromosome condensation 1	NM_001269	1853-2152
CD8 antigen, beta polypeptide 1 (p37)	NM_004931	155-454
B-cell CLL/lymphoma 7B	NM_001707	686-985
G protein-coupled receptor kinase 6	NM_002082	1483-1782
malate dehydrogenase 1, NAD (soluble)	NM_005917	713-1012
estrogen receptor 1	NM_000125	6059-6358
PCTAIRE protein kinase 3	AL161977	2041-1742
transcription factor AP-2 gamma	U85658	1898-2197
(activating enhancer-binding
protein 2 gamma)
signal transducer and activator	NM_003150	2380-2679
of transcription 3 (acute-phase
response factor)
mitogen-activated protein kinase	NM_005922	4978-5277
kinase kinase 4
CD9 antigen (p24)	NM_001769	556-855
lymphocyte-specific protein tyrosine	NM_005356	1361-1660
kinase
mutS (E. coli) homolog 3	NM_002439	635-934
mutS (E. coli) homolog 3	NM_002439	634-933
cadherin 6, type 2, K-cadherin	NM_004932	2624-2923
(fetal kidney)
membrane-associated tyrosine-	NM_004203	1594-1893
and threonine-specific cdc2-
inhibitory kinase
interferon regulatory factor 1	NM_002198	1558-1857
branched chain keto acid	NM_000709	852-1151
dehydrogenase E1, alpha
polypeptide (maple syrup urine disease)
mitogen-activated protein kinase 4	NM_002747	3570-3869

[0137]

TABLE 30
	Accession	Selected Gene
Name of Gene	Number	Region
ESTs, Highly similar to	BF568451	74-373
beta-1, 3-N-acetylgluco-
saminyltransferase
[H. sapiens]
cyclin-dependent kinase 5	NM_004935	609-908
hsp70-interacting protein	NM_012267	1185-1484
polo (Drosophia)-like	NM_005030	1384-1683
kinase
ninjurin 1	NM_004148	937-1236
thyroid receptor inter-	NM_004236	1102-1401
acting protein 15
LPS-induced TNF-alpha factor	NM_004862	458-757
fibroblast growth factor re-	M64347	3202-3501
ceptor 3 (achondroplasia,
thanatophoric dwarfism)
c-mer proto-oncogene tyrosine	NM_006343	2993-3292
kinase
carcinoembryonic antigen-	X16354	3053-3352
related cell adhesion mole-
cule 1 (biliary glycoprotein)
damage-specific DNA binding	NM_000107	492-791
protein 2 (48 kD)
damage-specific DNA binding	NM_000107	491-790
protein 2 (48 kD)
insulin-like growth factor	NM_002178	589-888
binding protein 6
Ras-related GTP-binding	NM_006570	1051-1350
protein
small inducible cytokine B	NM_006419	404-703
subfamily(Cys-X-Cys motif),
member 13 (B-cell chemo-
attractant)
autocrine motility factor	AF124145	2066-2365
receptor
thrombopoietin(myelopro-	NM_000460	1205-1504
liferative leukemia virus
oncogene ligand, mega-
karyocyte growth and
development factor)

[0138]

TABLE 31
	Accession	Selected Gene
Name of Gene	Number	Region
prostaglandin E synthase	AF217965	1519-1818
integrin, alpha M (complement	NM_000632	3572-3871
component receptor 3, alpha; also
known as CD11b (p170), macrophage
antigen alpha polypeptide)
integrin, alpha M (complement component	NM_000632	3571-3870
receptor 3, alpha; also known as CD11b
(p170), macrophage antigen alpha
polypeptide)
met proto-oncogene (hepatocyte	NM_000245	790-1089
growth factor receptor)
37 kDa leucine-rich repeat	NM_005824	1402-1701
(LRR) protein
ubiquitin specific protease 9, X	NM_004652	7854-8153
chromosome (Drosophila fat facets
related)
uridine monophosphate synthetase	NM_000373	601-900
(orotate phosphoribosyl transferase
and orotidine-5′-decarboxylase)
uridine monophosphate synthetase	NM_000373	600-899
(orotate phosphoribosyl
transferase and orotidine-5′-
decarboxylase)
zona pellucida glycoprotein 3A	NM_007155	880-1179
(sperm receptor)
phosphorylase kinase, beta	NM_000293	3265-3564

[0139]

TABLE 32
	Accession	Selected Gene
Name of Gene	Number	Region
prefoldin 5	NM_002624	542-841
reticulon 3	NM_006054	791-1090
dual-specificity tyrosine-(Y)-	NM_006482	1393-1692
phosphorylation regulated kinase 2
interleukin 18 receptor 1	NM_003855	2830-3129
villin 2 (ezrin)	NM_003379	2744-3043
CDC16 (cell division cycle 16,	NM_003903	1735-2034
S. cerevisiae , homolog)
integrin, alpha 3 (antigen CD49C,	NM_002204	3756-4055
alpha 3 subunit of VLA-3 receptor)
integrin, alpha 3 (antigen CD49C,	NM_002204	3755-4054
alpha 3 subunit of VLA-3 receptor)
retinoic acid receptor, beta	NM_000965	1997-2296
non-metastatic cells 2, protein	NM_002512	342-641
(NM23B) expressed in
dystroglycan 1 (dystrophin-	NM_004393	4389-4688
associated glycoprotein 1)
Ras homolog enriched in brain 2	NM_005614	688-987
tyrosine kinase 2	NM_003331	3583-3882
branched chain aminotransferase 2,	NM_001190	1207-1506
mitochondrial
ephrin-B1	NM_004429	2213-2512
thrombospondin 4	NM_003248	2709-3008

[0140]

TABLE 33
	Accession	Selected Gene
Name of Gene	Number	Region
Human DNA sequence from clone 223H9	AL008582	80-379
on chromosome 22q12.3-13.2.
Contains the gene for TOB4 (BTG1
family protein) and the gene for
ortholog of A. thaliana F23F1.8.
Contains ESTs, STSs, GSSs and four
putative CpG islands
GTP-binding protein overexpressed	NM_005261	1320-1619
in skeletal muscle
bone morphogenetic protein 6	NM_001718	2347-2646
dihydrofolate reductase	NM_000791	2728-3027
E2F transcription factor 1	NM_005225	1862-2161
mitogen-activated protein kinase	L11285	1355-1056
kinase 2
glutathione transferase zeta 1	NM_001513	499-798
(maleylacetoacetate isomerase)
nucleoside phosphorylase	NM_000270	857-1156
collagen, type IV, alpha 2	AK025912	2644-2345
paxillin	NM_002859	2855-3154
Fanconi anemia, complementation	NM_004629	2228-2527
group G
CD34 antigen	NM_001773	1945-2244
CD34 antigen	NM_001773	1944-2243
integrin, beta 5	NM_002213	2588-2887
dihydropyrimidine dehydrogenase	NM_000110	3815-4114
Rho GTPase activating protein 1	U02570	3045-3344
Rho GTPase activating protein 1	U02570	3044-3343

[0141]

TABLE 34
	Accession	Selected Gene
Name of Gene	Number	Region
v-rel avian reticuloendo-	NM_021975	1998-2297
theliosis viral oncogene
homolog A (nuclear factor
of kappa light polypeptide
gene enhancer in B-cells
3 (p65))
v-rel avian reticuloendo-	NM_021975	1997-2296
theliosis viral oncogene
homolog A (nuclear factor
of kappa light polypeptide
gene enhancer in B-cells
3 (p65))
protocadherin 1 (cadherin-	NM_002587	3740-4039
like 1)
mitogen-activated protein	NM_001315	3179-3478
kinase 14
keratin 10 (epidermolytic	NM_000421	1630-1929
hyperkeratosis; keratosis
palmaris et plantaris)
serine/threonine kinase	NM_003576	1361-1660
24(Ste20, yeast homolog)
5T4 oncofetal trophoblast	NM_006670	1259-1558
glycoprotein
formyl peptide receptor 1	NM_002029	581-880
cadherin 3, type 1, P-cad-	NM_001793	2857-3156
herin (placental)
integrin, alpha E (antigen	NM_002208	3413-3712
CD103, human mucosal lympho-
cyte antigen 1; alpha poly-
peptide)
deleted in lymphocytic leu-	NM_005887	213-512
kemia, 1
protein tyrosine phosphatase,	NM_002828	987-1286
non-receptor type 2
retinoblastoma-binding pro-	NM_005610	1329-1628
tein 4
Fas (TNFRSF6)-associated via	NM_003824	1343-1642
death domain
glutathione-S-transferase	NM_004832	408-707
like; glutathione trans-
ferase omega

[0142]

TABLE 35
	Accession	Selected Gene
Name of Gene	Number	Region
neural precursor cell expressed,	D42055	5168-5467
developmentally down-regulated 4
catenin (cadherin-associated protein),	NM_001904	2635-2934
beta 1 (88 kD)
catenin (cadherin-associated protein),	NM_001904	2634-2933
beta 1 (88 kD)
suppression of tumorigenicity 5	NM_005418	3977-4276
ATP-binding cassette, sub-family C	NM_020038	5065-5364
(CFTR/MRP), member 3
adenylosuccinate synthase	AK025514	1199-900
caspase 8, apoptosis-related cysteine	X98172	2545-2844
protease
caspase 8, apoptosis-related cysteine	X98172	2546-2845
protease
v-erb-b2 avian erythroblastic leukemia	NM_004448	3790-4089
viral oncogene homolog 2 (neuro/
glioblastoma derived oncogene homolog)
tyrosine kinase with immunoglobulin	NM_005424	3388-3687
and epidermal growth factor homology
domains
fibroblast growth factor receptor 4	NM_002011	2400-2699
stromal cell-derived factor 1	NM_000609	2959-3258
matrix metalloproteinase 15	NM_002428	3125-3424
(membrane-inserted)
A kinase (PRKA) anchor protein	NM_005100	6230-6529
(gravin) 12
interferon gamma receptor 2	NM_005534	1475-1774
(interferon gamma transducer 1)

[0143]

	TABLE 36
		Accession	Selected Gene
	Name of Gene	Number	Region
	signal sequence receptor,	NM_003144	610-909
	alpha (translocon-associated
	protein alpha)
	GS2 gene	U03886	465-764
	ubiquitin-conjugating	NM_003969	288-587
	enzyme E2M (homologous to
	yeast UBC12)
	cell division cycle 25B	NM_021874	2670-2969
	Human DNA sequence from	AL157902	5521-5820
	clone RP4-675C20 on chro-
	mosome 1p13.2. Contains
	the 3′ end of the MAN1A2
	gene for mannosidase alpha
	1A2, a pseudogene similar
	to predicted fly, worm and
	yeast genes, ESTs, STSs
	and GSSs
	dishevelled 2 (homologous	NM_004422	287-586
	to Drosophila dsh)
	dishevelled 2 (homologous	NM_004422	286-585
	to Drosophila dsh)
	ataxia telangiectasia and	NM_001184	7656-7955
	Rad3 related
	tumor necrosis factor receptor	AF016266	1359-1658
	superfamily, member 10b
	cullin 2	NM_003591	2150-2449
	dual-specificity tyrosine-	AF263541	1421-1720
	(Y)-phosphorylation regu-
	lated kinase 4
	H2A histone family, member	NM_003512	1192-1491
	L
	proliferating cell nuclear	NM_002592	27-326
	antigen
	nuclear factor of kappa	NM_003998	3264-3563
	light polypeptide gene
	enhancer in B-cells 1 (p105)

[0144]

TABLE 37
	Accession	Selected Gene
Name of Gene	Number	Region
thioredoxin reductase 1	NM_003330	3103-3402
cyclin H	NM_001239	418-717
superoxide dismutase 3,	NM_003102	1586-1885
extracellular
guanine nucleotide binding	NM_004125	316-615
protein 10
testis-specific kinase 1	NM_006285	2124-2423
retinoblastoma-binding protein 2	NM_005056	5907-6206
growth arrest and DNA-damage-	NM_014330	1843-2142
inducible 34
growth arrest and DNA-damage-	NM_014330	1844-2143
inducible 34
growth arrest-specific 6	NM_000820	1591-1890
dystrophin (muscular dystrophy,	NM_004010	12818-13117
Duchenne and Becker types),
includes DXS142, DXS164, DXS206,
DXS230, DXS239, DXS268, DXS269,
DXS270, DXS272
adducing 3 (gamma)	NM_016824	2352-2651
ornithine decarboxylase 1	NM_002539	1224-1523
baculoviral IAP repeat-containing	NM_001168	965-1264
5 (survivin)
baculoviral IAP repeat-containing	NM_001168	964-1263
5 (survivin)
bone morphogenetic protein 4	NM_001202	1621-1920
bone morphogenetic protein 4	NM_001202	1620-1919
BCL2-like 2	D87461	2638-2937
ribosomal protein S5	NM_001009	1-300
interleukin16(lymphocyte chemoattractant	M90391	1904-2203
factor)

[0145]

TABLE 38
	Accession	Selected Gene
Name of Gene	Number	Region
plasminogen activator, urokinase	NM_002659	899-1198
receptor
defender against cell death 1	NM_001344	15-314
pleckstrin homology, Sec7 and	U59752	672-971
coiled/coil domains 2-like
keratin 19	NM_002276	645-944
deoxythymidylate kinase (thymidylate	NM_012145	593-892
kinase)
ephrin-B3	NM_001406	2694-2993
integrin, beta 4	NM_000213	4125-4424
Sjogren's syndrome/scleroderma	NM_006396	40-339
autoantigen 1
phosphoinositide-3-kinase, class 3	NM_002647	2610-2909
death-associated protein	NM_004394	1309-1608
absent in melanoma 2	NM_004833	113-412
mucin 1, transmembrane	J05582	3467-3766
mucin 1, transmembrane	J05582	3466-3765
signal transducer and activator of	NM_007315	3102-3401
transcription 1, 91 kD
alpha-2-macroglobulin	NM_000014	4172-4471
aldolase A, fructose-bisphosphate	NM_000034	300-599
aldolase A, fructose-bisphosphate	NM_000034	299-598
X-ray repair complementing defective	NM_021141	1721-2020
repair in Chinese hamster cells 5
(double-strand-break rejoining; Ku
autoantigen, 80 kD)

[0146]

TABLE 39
	Accession	Selected Gene
Name of Gene	Number	Region
X-ray repair complementing	NM_021141	1720-2019
defective repair in Chinese
hamster cells 5 (double-strand-
break rejoining; Ku autoantigen,
80 kD)
serine/threonine kinase 3 (Ste20,	NM_006281	2080-2379
yeast homolog)
thrombospondin 1	NM_003246	1780-2079
low density lipoprotein-related	NM_002332	13836-14135
protein 1 (alpha-2-macroglobulin
receptor)
B-cell associated protein	NM_007273	678-977
ankyrin 1, erythrocytic	NM_000037	6276-6575
myeloid leukemia factor 2	NM_005439	1083-1382
claudin 10	NM_006984	513-812
CDC37 (cell division cycle 37,	NM_007065	1087-1386
S. cerevisiae , homolog)
CDC37 (cell division cycle 37,	NM_007065	1088-1387
S. cerevisiae , homolog)
CDC28 protein kinase 1	NM_001826	80-379
glutathione peroxidase 1	NM_000581	631-930
adenosine monophosphate	AK025706	3707-3408
deaminase 2 (isoform L)
thyroid hormone receptor	AF000974	904-1203
interactor 6
F-box only protein 9	AL137520	1571-1870
lymphotoxin beta receptor	NM_002342	1563-1862
(TNFR superfamily, member 3
lymphotoxin beta receptor	NM_002342	1564-1863
(TNFR superfamily, member 3

[0147]

TABLE 40
	Accession	Selected Gene
Name of Gene	Number	Region
adenomatosis polyposis coli	NM_000038	8168-8467
glycophorin B (includes Ss	NM_002100	88-387
blood group)
mannosidase, alpha, class 1A,	NM_006699	577-876
member 2
KiSS-1 metastasis-suppressor	NM_002256	213-512
KiSS-1 metastasis-suppressor	NM_002256	212-511
H2B histone family, member Q	NM_003528	1426-1725
developmentally regulated GTP-	NM_001388	845-1144
binding protein 2
TNF receptor-associated factor 6	NM_004620	1635-1934
insulin-like growth factor 1	X57025	418-717
(somatomedin C)
coagulation factor II (thrombin)	NM_001992	2617-2916
receptor
ATP-binding cassette, sub-family	NM_000927	4183-4482
B (MDR/TAP), member 1
diaphorase(NADH/NADPH)(cytochrome	NM_000903	2142-2441
b-5 reductase)
proprotein convertase subtilisin/	NM_004716	2972-3271
kexin type 7
seven in absentia (Drosophila)	NM_003031	186-485
homolog 1
hepatoma-derived growth factor	NM_004494	1962-2261
(high-mobility group protein 1-like)
ATP synthase, H+ transporting,	NM_001688	780-1079
mitochondrial F0 complex, subunit
b, isoform 1
CASP8 and FADD-like apoptosis	Y14039	668-967
regulator
lumican	NM_002345	1239-1538
E74-like factor2(ets domain	NM_006874	2045-2344
transcription factor)
cytochrome c-1	NM_001916	492-791

[0148]

TABLE 41
	Accession	Selected Gene
Name of Gene	Number	Region
plasminogen activator, tissue	NM_000930	2153-2452
histone deacetylase 1	NM_004964	1646-1945
DEAD/H (Asp-Glu-Ala-Asp/His)	NM_004941	3465-3764
box polypeptide 8 (RNA helicase)
signaling lymphocytic activation	NM_003037	678-977
molecule
phorbol-12-myristate-13-acetate-	NM_021127	815-1114
induced protein1
serine protease inhibitor,	NM_021102	776-1075
Kunitz type, 2
BCL2-related protein A1	NM_004049	192-491
pleckstrin homology, Sec7 and	NM_004762	2708-3007
coiled/coil domains 1(cytohesin 1)
ancient ubiquitous protein 1	NM_012103	1348-1647
Rho guanine nucleotide exchange	NM_004706	2387-2686
factor (GEF) 1
lamin B2	M94362	3622-3921
v-myb avian myeloblastosis viral	NM_002466	1851-2150
oncogene homolog-like 2
lymphoid-restricted membrane protein	NM_006152	367-666
myxovirus (influenza) resistance 1,	NM_002462	2274-2573
homolog of murine (interferon-inducible
protein p78)
vitamin D (1,25-dihydroxyvitamin D3)	NM_000376	2860-3159
receptor
interferon-induced protein with	NM_001548	1200-1499
tetratricopeptide repeats 1
guanine nucleotide binding protein	NM_006496	1798-2097
(G protein), alpha inhibiting
activity polypeptide 3

[0149]

TABLE 42
	Accession	Selected Gene
Name of Gene	Number	Region
NIMA (never in mitosis	Z29067	1363-1662
gene a)-related kinase 3
UV radiation resistance	NM_003369	3145-3444
associated gene
zinc finger protein 173	NM_003449	3163-3462
potassium intermediate/	NM_002249	2027-2326
small conductance calcium-
activated channel, subfamily
N, member 3
zinc finger protein homologous	D89859	1381-1680
to Zfp161 in mouse
guanylate kinase 1	NM_000858	91-390
cell division cycle 2-like 5	AJ297709	4791-5090
(cholinesterase-related
cell division controller)
profilin 1	NM_005022	145-444
tumor protein p53-binding protein, 1	NM_005657	5803-6102
3-hydroxymethyl-3-methylglutaryl-	NM_000191	80-379
Coenzyme A lyase
(hydroxymethylglutaricaciduria)
3-hydroxymethyl-3-methylglutaryl-	NM_000191	79-378
Coenzyme A lyase
(hydroxymethylglutaricaciduria)
putative DNA/chromatin binding motif	AJ243706	6087-6386
collagen, type I, alpha 2	NM_000089	4389-4688
hexokinase 1	NM_000188	3191-3490
G-rich RNA sequence binding factor 1	NM_002092	1972-2271
vimentin	NM_003380	537-836
serum/glucocorticoid regulated kinase	NM_005627	1539-1838
early growth response 1	NM_001964	2658-2957
glutathione S-transferase M4	NM_000850	563-862

[0150]

TABLE 43
	Accession	Selected Gene
Name of Gene	Number	Region
guanylate binding protein 1,	NM_002053	1602-1901
interferon-inducible, 67 kD
transforming growth factor,	D50683	4613-4912
beta receptor II (70-80 kD)
Rho GDP dissociation inhibitor	NM_001175	501-800
(GDI) beta
DNA fragmentation factor, 45 kD,	NM_004401	990-1289
alpha polypeptide
vitiligo-associated protein VIT-1	NM_018693	2623-2922
cyclin D1 (PRAD1: parathyroid	X59798	3750-4049
adenomatosis 1)
cyclin D1 (PRAD1: parathyroid	X59798	3751-4050
adenomatosis 1)
retinoblastoma-binding protein 2	NM_005056	2315-2614
Human BRCA2 region, mRNA	U57962	2152-1853
sequence CG018
low density lipoprotein-related	NM_002337	1193-1492
protein-associated protein 1
(alpha-2-macroglobulin receptor-
associated protein 1)
low density lipoprotein-related	NM_002337	1192-1491
protein-associated protein 1
(alpha-2-macroglobulin receptor-
associated protein 1)
thyroid autoantigen 70 kD	NM_001469	887-1186
(Ku antigen)
cyclin-dependent kinase 4	NM_000075	851-1150
G1 to S phase transition 1	NM_002094	1693-1992
serine (or cysteine) proteinase	M93056	1161-862
inhibitor, clade B (ovalbumin),
member 1

[0151]

TABLE 44
	Accession	Selected Gene
Name of Gene	Number	Region
tissue inhibitor of	NM_003255	578-877
metalloproteinase 2
proliferation-associated	NM_006191	1249-1548
2G4, 38 kD
developmentally regulated GTP-	NM_004147	667-966
binding protein 1
nucleolar phosphoprotein p130	D21262	3156-3455
mesothelin	NM_013404	1350-1649
minichromosome maintenance	X74794	2597-2896
deficient (S. cerevisiae) 4
signal transducing adaptor molecule	AK024383	3726-3427
(SH3 domain and ITAM motif) 1
ubiquitin-like 1 (sentrin)	NM_003352	612-911
myeloid cell leukemia sequence 1	AF198614	834-1133
(BCL2-related)
keratin 7	NM_005556	1167-1466
keratin 7	NM_005556	1166-1465
non-metastatic cells 1, protein	NM_000269	90-389
(NM23A) expressed in (NME1), mRNA
major histocompatibility complex,	NM_002116	970-1269
class I, A
CDC-like kinase 3	NM_003992	1276-1575
cyclin-dependent kinase inhibitor 1B	AY004255	1714-2013
(p27, Kip1)
keratin 18	NM_000224	705-1004
immunoglobulin heavy constant	Y14737	864-1163
gamma 3(G3m marker)
PTK2 protein tyrosine kinase 2	NM_005607	1245-1544
interferon, gamma-inducible protein 30	NM_006332	623-922

[0152]

TABLE 45
	Accession	Selected Gene
Name of Gene	Number	Region
ribonucleotide reductase	NM_001033	2333-2632
M1 polypeptide
methylenetetrahydrofolate	NM_005956	1818-2117
dehydrogenase (NADP + dependent),
methenyltetrahydrofolate cyclohydrolase,
formyltetrahydrofolate synthetase
matrix metalloproteinase 19	NM_002429	2455-2754
CDC10 (cell division cycle 10,	NM_001788	1254-1553
S. cerevisiae , homolog)
transforming growth factor,	NM_000358	2362-2661
beta-induced, 68 kD
Human mRNA for SB classII	X03100	781-1080
histocompatibility antigen
alpha-chain
guanine nucleotide binding protein	NM_002070	391-690
(G protein), alpha inhibiting
activity polypeptide 2
transforming growth factor	AK027071	3370-3071
beta-stimulated protein TSC-22
tumor susceptibility gene 101	NM_006292	664-963
laminin receptor 1 (67 kD,	NM_002295	23-322
ribosomal protein SA)
pM5 protein	NM_014287	3671-3970
ras homolog gene family, member A	NM_001664	823-1122
polypyrimidine tract binding	NM_002819	2560-2859
protein (heterogeneous nuclear
ribonucleoprotein I)
lactate dehydrogenase A	NM_005566	1077-1376
heat shock protein 75	NM_016292	1290-1589

[0153]

TABLE 46
	Accession	Selected Gene
Name of Gene	Number	Region
heat shock protein 75	NM_016292	1289-1588
collagen, type VII, alpha	NM_000094	8962-9261
1 (epidermolysis bullosa,
dystrophic, dominant and recessive)
adenine phosphoribosyltransferase	NM_000485	542-841
keratin 8	NM_002273	448-747
anti-oxidant protein 2 (non-	NM_004905	1354-1653
selenium glutathione peroxidase,
acidic calcium-independent
phospholipase A2)
connective tissue growth factor	NM_001901	1966-2265
insulin-like growth factor binding	M35878	3082-3381
protein 3
phosphoglycerate kinase 1	NM_000291	903-1202
ubiquitin-conjugating enzyme E2A	NM_003336	1393-1692
(RAD6 homolog)
superoxide dismutase 1, soluble	NM_000454	79-378
(amyotrophic lateral sclerosis 1
(adult))
cyclin A2	NM_001237	1025-1324
high-mobility group (nonhistone	NM_002128	562-861
chromosomal) protein 1
neurotrophic tyrosine kinase,	NM_002530	859-1158
receptor, type 3
EphB6	NM_004445	3658-3957
tyrosine 3-monooxygenase/tryptophan	NM_003406	232-531
5-monooxy-genase activation protein,
zeta polypeptide
alkylation repair; alkB homolog	NM_006020	1556-1855

[0154]

TABLE 47
	Accession	Selected Gene
Name of Gene	Number	Region
keratin 4	X07695	1335-1634
baculoviral IAP repeat-containing 1	NM_004536	4270-4569
nitric oxide synthase 2A(inducible,	NM_000625	3556-3855
hepatocytes)
nitric oxide synthase 2A(inducible,	NM_000625	3555-3854
hepatocytes)
p21/Cdc42/Rac1-activated kinase 1	NM_002576	1890-2189
(yeast Ste20-related)
elastase 1, pancreatic	NM_001971	286-585
elastase 3, pancreatic (protease E)	NM_005747	163-462
interferon induced transmembrane	NM_006435	606-905
protein 2(1-8 D)
interferon induced transmembrane	NM_006435	605-904
protein 2(1-8 D)
phospholipase A2, group IB (pancreas)	NM_000928	213-512
cadherin 18, type 2	NM_004934	2565-2864
Tubulin, alpha, brain-specific	NM_006009	917-1216
phospholipase A2, group VI	NM_003560	2954-3253
(cytosolic, calcium-independent)
collagen, type VIII, alpha 1	NM_001850	1765-2064
matrix metalloproteinase 2	NM_004530	2496-2795
(gelatinase A, 72 kD gelatinase,
72 kD type IV collagenase)
matrix metalloproteinase 2	NM_004530	2495-2794
(gelatinase A, 72 kD gelatinase,
72 kD type IV collagenase)
microtubule-associated protein,	NM_012325	1469-1768
RP/EB family, member 1
interleukin 1, alpha	M28983	1211-1510

[0155]

TABLE 48
	Accession	Selected Gene
Name of Gene	Number	Region
eukaryotic translation initiation	NM_003757	585-884
factor 3, subunit 2 (beta, 36 kD)
galactosidase, beta 1	NM_000404	2020-2319
B cell RAG associated protein	NM_014863	3996-4295
B cell RAG associated protein	NM_014863	3995-4294
transforming growth factor, alpha	NM_003236	3569-3868
serine protease inhibitor,	NM_003710	1800-2099
Kunitz type 1
CD86 antigen(CD28 antigen ligand 2,	NM_006889	1069-1368
B7-2 antigen)
nuclear factor of kappa light	NM_004556	1365-1664
polypeptide gene enhancer in
B-cells inhibitor, epsilon
interferon, gamma	NM_000619	833-1132
O-6-methylguanine-DNA	NM_002412	266-565
methyltransferase
tyrosine 3-monooxygenase/tryptophan	NM_003406	1425-1724
5-monooxy-genase activation protein,
zeta polypeptide
Homo sapiens cDNA: FLJ20886 fis,	AK024539	25-324
clone ADKA03257
Homo sapiens cDNA: FLJ20886 fis,	AK024539	24-323
clone ADKA03257
BCL2/adenovirus E1B 19 kD-interacting	NM_013979	542-841
protein 1
DNAJ domain-containing	NM_013238	521-820
RAD51 (S. cerevisiae) homolog C	NM_002876	1-300
laminin, alpha 4	NM_002290	4992-5291
ESTs, Moderately similar to endothelial	BF666712	107-406
nitric oxide synthase [H. sapiens]

[0156]

TABLE 49
	Accession	Selected Gene
Name of Gene	Number	Region
ESTs, Moderately similar to	BF666712	106-405
endothelial nitric oxide synthase
[H. sapiens]
vascular endothelial growth factor	AF022375	2067-2366
growth differentiation factor 10	NM_004962	1509-1808
MAP/microtubule affinity-	NM_002376	1390-1689
regulating kinase 3
MAP/microtubule affinity-	NM_002376	1389-1688
regulating kinase 3
retinoic acid receptor, beta	NM_000965	1997-2296
cell division cycle 25B	NM_021874	1422-1721
erythropoietin receptor	NM_000121	617-916
erythropoietin receptor	NM_000121	616-915
immunoglobulin lambda locus	X57809	1-300
immunoglobulin lambda locus	X57809	2-301
fms-related tyrosine kinase 1	NM_002019	4007-4306
(vascular endothelial growth
factor/vascular permeability
factor receptor)
macrophage stimulating 1	AL137798	1237-1536
(hepatocyte growth factor-like)
macrophage stimulating 1	AL137798	1236-1535
(hepatocyte growth factor-like)
interleukin 1 receptor antagonist	U65590	1503-1802
KH-type splicing regulatory protein	NM_003685	1822-2121
(FUSE binding protein 2)

[0157]

TABLE 50
	Accession	Selected Gene
Name of Gene	Number	Region
KH-type splicing regulatory	NM_003685	1821-2120
protein (FUSE binding protein 2)
junction plakoglobin	NM_002230	2157-2456
junction plakoglobin	NM_002230	2156-2455
frizzled-related protein	NM_001463	627-926
frizzled-related protein	NM_001463	626-925
intercellular adhesion molecule	NM_000201	2421-2720
1 (CD54), human rhinovirus receptor
intercellular adhesion molecule	NM_000201	2420-2719
1 (CD54), human rhinovirus receptor
TGFB inducible early growth response	NM_005655	918-1217
jagged 2	NM_002226	1670-1969
jagged 2	NM_002226	1669-1968
cadherin, EGF LAG seven-pass	AF234887	9344-9643
G-type receptor 2, flamingo
(Drosophila) homolog
cadherin, EGF LAG seven-pass	AF234887	9345-9644
G-type receptor 2, flamingo
(Drosophila) homolog
signal transducer and activator	NM_003151	1212-1511
of transcription 4
matrix metalloproteinase	NM_002426	604-903
12(macrophage elastase)
glycogen synthase kinase 3 beta	NM_002093	98-397
glycogen synthase kinase 3 beta	NM_002093	99-398
eukaryotic translation initiation	NM_003908	990-1289
factor 2, subunit 2 (beta, 38 kD)

[0158]

TABLE 51
	Accession	Selected Gene
Name of Gene	Number	Region
disabled (Drosophila)	NM_001343	988-1287
homolog 2 (mitogen-responsive
phosphoprotein)
disabled (Drosophila)	NM_001343	989-1288
homolog 2 (mitogen-responsive
phosphoprotein)
BTG family, member 2	NM_006763	3-302
nuclear domain 10 protein	NM_005831	541-840
FYN oncogene related to SRC,	Z97989	331-630
FGR, YES
phosphatidylinositol glycan,	NM_002643	509-808
class F
phosphatidylinositol glycan,	NM_002643	508-807
class F
RAB36, member RAS oncogene	NM_004914	3377-3676
family
ESTs	N38956	378-79
protein phosphatase 1D magnesium-	NM_003620	1656-1955
dependent, delta isoform
dihydrofolate reductase	NM_000791	1537-1836
ubiquitin-activating enzyme E1-like	NM_003335	2533-2832
fibronectin 1	X02761	141-440
hepatocyte growth factor-regulated	NM_004712	1713-2012
tyrosine kinase substrate
transcription factor Dp-2 (E2F	NM_006286	1-300
dimerization partner 2)
transcription factor Dp-2 (E2F	NM_006286	2-301
dimerization partner 2)
mutS (E. coli) homolog 6	NM_000179	2750-3049
DNA (cytosine-5-)-methyltransferase 1	NM_001379	4606-4905

[0159]

TABLE 52
	Accession	Selected Gene
Name of Gene	Number	Region
DNA (cytosine-5-)-methyltransferase 1	NM_001379	4605-4904
MAD(mothers against decapentaplegic,	NM_005901	1596-1895
Drosophila ) homolog 2
fms-related tyrosine kinase 1	NM_002019	7381-7680
(vascular endothelial growth
factor/vascular permeability
factor receptor)
CD22 antigen	NM_001771	2273-2572
CD22 antigen	NM_001771	2272-2571
folate hydrolase (prostate-	NM_004476	1694-1993
specific membrane antigen) 1
jun D proto-oncogene	NM_005354	1271-1570
Human glucocorticoid receptor	U25029	1282-1581
alpha mRNA, variant 3′ UTR
laminin, alpha 4	NM_002290	5481-5780
nuclear factor of kappa light	NM_020529	1204-1503
polypeptide gene enhancer in
B-cells inhibitor, alpha
ClpP (caseinolytic protease,	NM_006012	513-812
ATP-dependent, proteolytic
subunit, E. coli) homolog
frizzled-related protein	NM_001463	627-926
frizzled-related protein	NM_001463	626-925
linker for activation of T cells	NM_014387	138-437
guanine nucleotide binding protein	NM_000516	26-325
(G protein), alpha stimulating
activity polypeptide 1
guanine nucleotide binding protein	NM_000516	25-324
(G protein), alpha stimulating
activity polypeptide 1

[0160]

TABLE 53
	Accession	Selected Gene
Name of Gene	Number	Region
fibroblast growth factor 1 (acidic)	X59065	1599-1898
retinoblastoma-binding protein 6	NM_006910	1754-2053
high-mobility group (nonhistone	NM_002129	474-773
chromosomal) protein 2
high-mobility group (nonhistone	NM_002129	473-772
chromosomal) protein 2
death-associated protein kinase 1	NM_004938	5463-5762
cysteine-rich, angiogenic inducer, 61	NM_001554	1211-1510
keratin 1 (epidermolytic hyperkeratosis)	NM_006121	1871-2170
fms-related tyrosine kinase 1	NM_002019	6664-6963
(vascular endothelial growth factor/
vascular permeability factor receptor)
heterogeneous nuclear ribonucleoprotein K	NM_002140	1-300
triple functional domain	NM_007118	7588-7887
(PTPRF interacting)
adenomatosis polyposis coli	NM_000038	8168-8467
BRCA1 associated RING domain 1	NM_000465	1669-1968
BRCA1 associated RING domain 1	NM_000465	1668-1967
granulysin	NM_012483	484-783
dishevelled 3 (homologous to	NM_004423	2100-2399
Drosophila dsh)
splicing factor, arginine/serine-	NM_004592	2785-3084
rich 8(suppressor-of-white-apricot,
Drosophila homolog)

[0161]

TABLE 54
	Accession	Selected Gene
Name of Gene	Number	Region
polymerase (DNA directed), alpha	NM_016937	4915-5214
tec protein tyrosine kinase	NM_003215	1881-2180
H2B histone family, member Q	NM_003528	1191-1490
seven in absentia (Drosophila)	U76248	1564-1863
homolog 2
coronin, actin-binding protein, 1A	NM_007074	179-478
uridine phosphorylase	NM_003364	987-1286
adenosine deaminase	NM_000022	1180-1479
growth arrest and DNA-damage-	L24498	2224-2523
inducible, alpha
receptor (TNFRSF)-interacting	NM_003804	729-1028
serine-threonine kinase 1
receptor (TNFRSF)-interacting	NM_003804	728-1027
serine-threonine kinase 1
transforming growth factor, beta 2	NM_003238	92-391
transforming growth factor, beta 2	NM_003238	91-390
zinc finger protein, subfamily 1A,	NM_006060	2309-2608
1 (Ikaros)
translocated promoter region	NM_003292	6604-6903
(to activated MET oncogene)
serine/threonine kinase 25	NM_006374	1469-1768
(Ste20, yeast homolog)
solute carrier family 2	NM_006931	1598-1897
(facilitated glucose
transporter), member 3
solute carrier family 2	NM_006931	1599-1898
(facilitated glucose
transporter), member 3
ESTs, Highly similar to neuronal	AI821682	381-82
apoptosis inhibitory protein
[H. sapiens]

[0162]

TABLE 55
	Accession	Selected Gene
Name of Gene	Number	Region
basigin	NM_001728	708-1007
basigin	NM_001728	707-1006
cyclin-dependent kinase 5,	NM_003885	322-621
regulatory subunit 1 (p35)
cyclin-dependent kinase 5,	NM_003885	323-622
regulatory subunit 1 (p35)
X-ray repair complementing	NM_006297	1720-2019
defective repair in
Chinese hamster cells 1
tyrosinase-related protein 1	NM_000550	1296-1595
Pseudoautosomal GTP-binding	NM_012227	1557-1856
protein-like
Pseudoautosomal GTP-binding	NM_012227	1556-1855
protein-like
antigen identified by monoclonal	X65550	12195-12494
antibody Ki-67
antigen identified by monoclonal	X65550	12194-12493
antibody Ki-67
neurofibromin 2 (bilateral acoustic	NM_000268	1899-2198
neuroma)
neurofibromin 2 (bilateral acoustic	NM_000268	1898-2197
neuroma)
nucleotide binding protein 2 (E. coli	NM_012225	485-784
MinD like)
nucleotide binding protein 2 (E. coli	NM_012225	484-783
MinD like)
solute carrier family 25 (mitochondrial	NM_001152	839-1138
carrier; adenine nucleotide translocator),
member 5
laminin, beta 1	NM_002291	4820-5119
laminin, beta 1	NM_002291	4821-5120
seven in absentia (Drosophila) homolog 1	NM_003031	117-416

[0163]

TABLE 56
	Accession	Selected Gene
Name of Gene	Number	Region
high-mobility group (nonhistone	NM_002131	1196-1495
chromosomal) protein isoforms
I and Y
BCL2/adenovirus E1B 19 kD-interacting	NM_004052	313-612
protein 3
D123 gene product	NM_006023	1015-1314
prostate differentiation factor	NM_004864	286-585
interferon regulatory factor 3	NM_001571	543-842
interferon regulatory factor 3	NM_001571	544-843
uroporphyrinogen III synthase	NM_000375	730-1029
(congenital erythropoietic
porphyria)
uroporphyrinogen III synthase	NM_000375	731-1030
(congenital erythropoietic
porphyria)
minichromosome maintenance	NM_004526	2969-3268
deficient (S. cerevisiae)
2 (mitotin)
collagen, type VI, alpha 2	AL096746	1345-1644
restin (Reed-Steinberg cell-	NM_002956	4668-4967
expressed intermediate filament-
associated protein)
restin (Reed-Steinberg cell-	NM_002956	4667-4966
expressed intermediate filament-
associated protein)
v-fos FBJ murine osteosarcoma	NM_005252	60-359
viral oncogene homolog
v-fos FBJ murine osteosarcoma	NM_005252	59-358
viral oncogene homolog
tyrosine 3-monooxygenase/tryptophan	NM_003406	232-531
5-monooxy-genase activation protein,
zeta polypeptide

[0164]

TABLE 57
	Accession	Selected Gene
Name of Gene	Number	Region
glutathione synthetase	NM_000178	1483-1782
retinoid X receptor, alpha	NM_002957	4358-4657
tumor necrosis factor, alpha-	NM_021137	1237-1536
induced protein 1 (endothelial)
tumor necrosis factor, alpha-	NM_021137	1236-1535
induced protein 1 (endothelial)
GS3955 protein	NM_021643	3692-3991
MAD2 (mitotic arrest deficient,	NM_002358	125-424
yeast, homolog)-like 1
A kinase (PRKA) anchor protein 1	NM_003488	2434-2733
vaccinia related kinase 2	NM_006296	1010-1309
neutrophil cytosolic factor 1	NM_000265	322-621
(47 kD, chronic granulomatous disease,
autosomal 1)
RAD23 (S. cerevisiae) homolog B	NM_002874	2302-2601
postmeiotic segregation increased	NM_000534	2327-2626
(S. cerevisiae) 1
vinculin	NM_014000	4722-5021
karyopherin alpha 3 (importin alpha 4)	NM_002267	1802-2101
thymidylate synthetase	NM_001071	1088-1387
thymidylate synthetase	NM_001071	1087-1386
chitinase 3-like 2	NM_004000	516-815
protein kinase, cGMP-dependent, type I	NM_006258	2516-2815

[0165]

TABLE 58
	Accession	Selected Gene
Name of Gene	Number	Region
nuclear factor of activated T-cells,	U80917	4122-4421
cytoplasmic, calcineurin-dependent 1
heat shock 70 kD protein 4	AB023420	2381-2680
proline-rich protein with nuclear	NM_006813	885-1184
targeting signal
nuclear factor, interleukin 3	NM_005384	810-1109
regulated
quinone oxidoreductase homolog	NM_004881	1129-1428
actin, alpha 2, smooth muscle, aorta	NM_001613	1-300
actin, alpha 2, smooth muscle, aorta	NM_001613	2-301
Fc fragment of IgG, low affinity IIa,	NM_021642	1707-2006
receptor for (CD32)
sodium bicarbonate transporter 4	NM_021196	921-1220
sodium bicarbonate transporter 4	NM_021196	922-1221
ras-related C3 botulinum toxin	NM_018890	62-361
substrate 1 (rho family, small GTP
binding protein Rac1)
ras-related C3 botulinum toxin	NM_018890	61-360
substrate 1 (rho family, small GTP
binding protein Rac1)
dual-specificity tyrosine-(Y)-	D86550	5323-5622
phosphorylation regulated kinase 1A
catenin (cadherin-associated protein),	NM_001903	2813-3112
alpha 1 (102 kD)
catenin (cadherin-associated protein),	NM_001903	2812-3111
alpha 1 (102 kD)
v-abl Abelson murine leukemia	NM_005157	4953-5252
viral oncogene homolog 1

[0166]

TABLE 59
	Accession	Selected Gene
Name of Gene	Number	Region
v-abl Abelson murine leukemia	NM_005157	4952-5251
viral oncogene homolog 1
tousled-like kinase 2	NM_006852	2205-2504
protein tyrosine phosphatase,	NM_002840	7099-7398
receptor type, F
protein tyrosine phosphatase,	NM_002840	7098-7397
receptor type, F
osteoblast specific factor 2	NM_006475	1889-2188
(fasciclin I-like)
preferentially expressed	NM_006115	1743-2042
antigen in melanoma
phosphoribosylglycinamide	NM_000819	2539-2838
formyltransferase, phos-
phoribosylglycinamide
synthetase, phos-
phoribosylaminoimidazole
synthetase
early development regulator 2	NM_004427	1897-2196
(homolog of polyhomeotic 2)
hypothetical protein FLJ10262	AB037730	4530-4829
bone morphogenetic protein 5	NM_021073	1452-1751
bone morphogenetic protein8	NM_001720	1114-1413
(osteogenic protein2)
bone morphogenetic protein8	NM_001720	1113-1412
(osteogenic protein2)
cyclin-dependent kinase in-	NM_000077	1-300
hibitor 2A (melanoma, p16,
inhibits CDK4)
cyclin-dependent kinase in-	NM_000077	2-301
hibitor 2A (melanoma, p16,
inhibits CDK4)
POU domain, class 2, tran-	NM_002698	1281-1580
scription factor 2
amphiregulin (schwannoma-	NM_001657	80-379
derived growth factor)

[0167]

TABLE 60
	Accession	Selected Gene
Name of Gene	Number	Region
amphiregulin (schwannoma-derived	NM_001657	79-378
growth factor)
cytochrome c	BF214508	1-300
proteasome (prosome, macropain)	NM_002800	376-675
subunit, beta type, 9 (large
multifunctional protease 2)
ephrin-A1	NM_004428	1-300
RAD51(S. cerevisiae)homolog(E coli	NM_002875	499-798
RecA homolog)
small inducible cytokine subfamily	NM_001565	431-730
B(Cys-X-Cys), member 10
CDC16 (cell division cycle 16,	NM_003903	24-323
S. cerevisiae , homolog)
ras-like protein	NM_012249	135-434
ras-like protein	NM_012249	134-433

[0168] In order to prepare a DNA chip on which each of the nucleic acids having a nucleotide sequence of a selected gene region was spotted, an amplified nucleic acid fragment of the gene region was first prepared.

[0169] A primer of 20 bases corresponding to an upstream sequence of the specific gene region of each of the genes listed in Tables 4 to 60 and a primer of 20 bases corresponding to a downstream sequence thereof were synthesized using a DNA synthesizer (manufactured by Bio Automation), to give each of primer pairs. PCR was carried out by a conventional method using each of the above primer pairs, to give a desired amplified DNA fragment. Each of the resulting amplified DNA fragments was purified using Qiaquick PCR purification kit 96 (manufactured by QIAGEN) in accordance with the attached protocol.

[0170] Each of the above amplified fragments was purified, and thereafter spotted on each of the above slide glass into which an activated carboxyl group was introduced and on TaKaRa Slide Glass (manufactured by Takara Bio Inc.) using the Affymetrix 417 Arrayer (manufactured by Affymetrix).

[0171] The slide glass after spotting, into which an activated carboxyl group was introduced was washed with 0.2% SDS, and then washed twice with distilled water. Thereafter, the above slide glass was treated with 0.3 N NaOH for 5 minutes, washed twice with distilled water, and dried by centrifugation (150× g for 2 minutes). On the other hand, TaKaRa Slide Glass after spotting was subjected to post-treatment in accordance with the instruction manual attached.

[0172] The preparation of the labeled cDNA probes, hybridization, washing and analysis were carried out under the same conditions as those described in Example 1.

[0173] The analytical results for the genes listed in Tables 4 to 60 in the combinations of the non-labeled substrate/labeled substrate for each of Cy3 and Cy5 are shown in Tables 61 and 62. Table 61 shows the results of a case where the DNA fragment was immobilized on the substrate by electrostatic bonding, i.e., a case where TaKaRa Slide Glass was used. Table 62 shows the results of a case where the DNA fragment was immobilized on the substrate by covalent bonding, i.e., a case where the slide glass into which an activated carboxyl group was introduced was used.

	TABLE 61
	Non-Labeled Substrate/	Cy5
	Labeled Substrate	5/1	3.5/1	2/1
		5/1	2.41
	Cy3	3.5/1	1.97	2.07
		2/1	1.56	1.94	2.57

[0174]

	TABLE 62
	Non-Labeled Substrate/	Cy5
	Labeled Substrate	5/1	3.5/1	2/1
		5/1	2.01
	Cy3	3.5/1	1.86	2.13
		2/1	1.53	1.70	2.11

[0175] As shown in Tables 61 and 62, similar results were obtained from both of slide glasses used. In other words, as shown in this example, it was suggested that there are not be influenced by a surface treatment of the substrate or by a binding manner between the nucleic acid to be immobilized and the substrate by using a concentration ratio for the non-labeled substrate/labeled substrate suitable for each of Cy3 and Cy5. In particular, it was found that the analytical results were the most favorable for both of the cases of TaKaRa Slide Glass and the slide glass into which an activated carboxyl group was introduced, when the concentration ratio of a non-labeled substrate/labeled substrate for Cy3 was 2/1 and when the concentration ratio of a non-labeled substrate/labeled substrate for Cy5 was 5/1.

EXAMPLE 4

[0176] The optimum concentration ratio of the non-labeled substrate/labeled substrate for Cy5 was evaluated by varying a concentration ratio of the non-labeled substrate/labeled substrate for Cy5 within the range of 3/1 to 9/1, with fixing a concentration ratio of the non-labeled substrate/labeled substrate for Cy3 at 2/1. Each of the substrate concentrations is shown in Table 63.

TABLE 63
Non-Labeled Substrate/Labeled Substrate = 3/1
dATP    0.20 mM
dGTP    0.20 mM
dCTP    0.20 mM
dTTP    0.15 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 5/1
dATP    0.30 mM
dGTP    0.30 mM
dCTP    0.30 mM
dTTP    0.25 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 7/1
dATP    0.40 mM
dGTP    0.40 mM
dCTP    0.40 mM
dTTP    0.35 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 9/1
dATP    0.50 mM
dGTP    0.50 mM
dCTP    0.50 mM
dTTP    0.45 mM
Cy-dUTP 0.05 mM

[0177] The preparation of the labeled cDNA probes, hybridization, washing and analytical conditions were carried under the same conditions as those described in Example 1. The studied combinations and the results are shown in Table 64.

	TABLE 64
	Non-Labeled Substrate/		Cy5
	Labeled Substrate		3/1	5/1	7/1	9/1
	Cy3	2.0	2.01	1.60	1.82	1.72

[0178] As shown in Table 64, when the concentration ratio of the non-labeled substrate/labeled substrate for Cy3 was fixed at 2/1, it was found that the results of high accuracy were obtained within any of the range of the concentration ratios of the non-labeled substrate/labeled substrate for Cy5 of 5/1 to 9/1, and that especially in a case where the concentration ratio was 5/1, the accuracy became the highest.

EXAMPLE 5

[0179] The optimum concentration ratio of the non-labeled substrate/labeled substrate for Cy3 was evaluated by varying a concentration ratio of the non-labeled substrate/labeled substrate for Cy3 within the range of 1/1 and 4/1 with fixing a concentration ratio of the non-labeled substrate/labeled substrate for Cy5 at 5/1. Each of the substrate concentrations is shown in Table 65.

TABLE 65
Non-Labeled Substrate/Labeled Substrate = 1/1
dATP    0.10 mM
dGTP    0.10 mM
dCTP    0.10 mM
dTTP    0.05 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 2/1
dATP    0.15 mM
dGTP    0.15 mM
dCTP    0.15 mM
dTTP    0.10 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 3/1
dATP    0.20 mM
dGTP    0.20 mM
dCTP    0.20 mM
dTTP    0.15 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 4/1
dATP    0.25 mM
dGTP    0.25 mM
dCTP    0.25 mM
dTTP    0.20 mM
Cy-dUTP 0.05 mM

[0180] The preparation of the labeled cDNA probes, hybridization, washing and analytical conditions were carried out under the same conditions as those described in Example 1. The studied combinations and the results are shown in Table 66.

	TABLE 66
	Non-Labeled Substrate/		Cy5
	Labeled Substrate		1/1	2/1	3/1	4/1
	Cy5	5/1	1.73	1.48	1.53	1.54

[0181] As shown in Table 66, when the concentration ratio of the non-labeled substrate/labeled substrate for Cy5 was fixed at 5/1, the concentration ratios of the non-labeled substrate/labeled substrate for Cy3 were preferably 2/1 or higher, and the results of high accuracy were obtained with any of the concentration ratios of 2/1, 3/1, and 4/1. In particular, it was found that it is preferable to set to have as low concentration ratio of the non-labeled substrate/labeled substrate for Cy3 as possible, in order to increase the ratio of significant spots, and it is particularly preferable to have the non-labeled substrate/labeled substrate of 2/1.

EXAMPLE 6

[0182] Studies were made on the method of the present invention with varying the kinds of reverse transcriptases. In this example, a reverse transcriptase derived from AMV (Avian myeloblastosis virus) (manufactured by Takara Bio Inc.) was used. Also, the concentration ratio for the non-labeled substrate/labeled substrate was set at 1.86/1 for both Cy3 and Cy5, or set at 2/1 for Cy3 and 5/1 for Cy5, and the analytical results were compared. Each of the substrate concentrations is shown in Table 67. The studied combinations of the concentration ratios of the fluorescent substance and the non-labeled substrate/labeled substrate and the results are shown in Table 68. Here, the preparation of the labeled cDNA probes, hybridization with the DNA microarray, washing, scanning and analysis were carried out under the same conditions as those described in Example 1.

TABLE 67
Non-Labeled Substrate/Labeled Substrate = 5/1
dATP    0.30 mM
dGTP    0.30 mM
dCTP    0.30 mM
dTTP    0.25 mM
Cy-dUTP 0.05 mM
Non-Labeled Substrate/Labeled Substrate = 1.86/1
dATP    0.1 mM
dGTP    0.1 mM
dCTP    0.1 mM
dTTP    0.065 mM
Cy-dUTP 0.035 mM
Non-Labeled Substrate/Labeled Substrate = 2/1
dATP    0.15 mM
dGTP    0.15 mM
dCTP    0.15 mM
dTTP    0.10 mM
Cy-dUTP 0.05 mM

[0183]

TABLE 68
Cy3	Cy5	Analytical
	Non-Labeled Substrate/Labeled Substrate	Accuracy
1.86/1	1.86/1	2.89
2.0/1	5.0/1	1.46

[0184] As shown in Table 68, when the concentration ratios of the non-labeled substrate/labeled substrate for both Cy3 and Cy5 were fixed at the same 1.86/1, a Cy3/Cy5 signal ratio at a 99% convergence in distribution of the significant spots (i.e., analytical accuracy) was shown to have a value of 2.89. When the concentration ratios of the non-labeled substrate/labeled substrate were fixed at 2/1 for Cy3 and 5/1 for Cy5, the Cy3/Cy5 signal ratio (i.e., analytical accuracy) became 1.46. Therefore, it was found that the improvement in the analytical accuracy was seen even when the AMV-derived reverse transcriptase was used.

[0185] In other words, it was found that as a result of gene expression analysis using a DNA chip or DNA microarray, the expression of all of the genes approximated 1:1 when the same mRNA of the same amount was labeled with each of the fluorescent markers Cy3 and Cy5 by setting the concentration ratio of the labeled substrate/non-labeled substrate separately for Cy3 and Cy5, even when the reverse transcriptase derived from AMV was used.

INDUSTRIAL APPLICABILITY

[0186] According to the labeling method of the present invention, there can be provided a labeled nucleic acid capable of performing analysis with reflecting the inherent profile of gene expression. Therefore, according to the labeling method of the present invention, there can be accurately grasped the behavior of the gene expressions in gene expression analysis. Also, according to the present invention, there can be provided a kit for fluorescent-labeling a probe capable of performing the gene expression analysis using the dual color hybridization method at high accuracy.

Claims

1. A method for labeling a nucleic acid, wherein the method is a method for labeling the nucleic acid with at least two kinds of different labeled substances distinguishable from each other, and wherein the method comprises the step of labeling the nucleic acid in a nucleic acid sample containing plural kinds of nucleic acids by use of: one labeled substrate which is labeled with a labeling substance and a non-labeled substrate corresponding thereto, in an amount ratio satisfying the following conditions that a ratio of: a) a signal intensity of a signal ascribed to a labeled nucleic acid prepared with the nucleic acid in the nucleic acid samples as a template, wherein the labeled nucleic acid is labeled with the labeled substrate, to b) a signal intensity of a signal ascribed to a labeled nucleic acid prepared with the same nucleic acid as that of the above a) as a template, wherein the labeled nucleic acid is labeled with a labeled substrate different from the labeled substrate of the above a) in each of the nucleic acids in the nucleic acid sample is substantially the same, irrelevant to the kinds of the nucleic acids to be used as a template.

2. The method according to claim 1, wherein the labeled nucleic acid is prepared by reverse transcription reaction from the nucleic acid used as a template.

3. The method according to claim 1 or 2, wherein the different labeled substrate is Cy3-labeled substrate or Cy5-labeled substrate.

4. The method according to claim 3, wherein the nucleic acid in the nucleic acid sample is labeled in a reaction mixture containing the non-labeled substrate and the Cy3-labeled substrate in a concentration ratio (the non-labeled substrate/the Cy3-labeled substrate) within the range of from 1/1 to 5/1.

5. The method according to claim 3 or 4, wherein the nucleic acid in the nucleic acid sample is labeled in a reaction mixture containing the non-labeled substrate and the Cy5-labeled substrate in a concentration ratio (the non-labeled substrate/the Cy5-labeled substrate) within the range of from 3/1 to 10/1.

6. A labeled nucleic acid prepared by the method of any one of claims 1 to 5.

7. A kit for labeling a nucleic acid comprising an instruction manual describing the procedures of the method of any one of claims 1 to 5.

8. The kit according to claim 7, wherein the instruction manual describes a method for preparing a mixed substrate containing the Cy3-labeled substrate and the non-labeled substrate corresponding thereto in a concentration ratio (the non-labeled substrate/the Cy3-labeled substrate) within the range of from 1/1 to 9/1.

9. The kit according to claim 7 or 8, wherein the instruction manual describes a method for preparing a mixed substrate containing the Cy5-labeled substrate and the non-labeled substrate corresponding thereto in a concentration ratio (the non-labeled substrate/the Cy5-labeled substrate) within the range of from 3/1 to 10/1.

10. A kit for labeling a nucleic acid, comprising: (1) a reaction vessel containing a reaction mixture in an amount of one-time use or defined times of use, wherein the reaction mixture comprises a Cy3-labeled substrate and a non-labeled substrate corresponding thereto in a concentration ratio (non-labeled substrate/Cy3-labeled substrate) of from 1/1 to 5/1, and comprises a non-labeled nucleotide substrate other than the above non-labeled substrate, and/or (2) a reaction vessel containing a reaction mixture in an amount for one-time use or defined times of use, wherein the reaction mixture comprises a Cy5-labeled substrate and a non-labeled substrate corresponding thereto in a concentration ratio (non-labeled substrate/Cy5-labeled substrate) within the range of from 3/1 to 10/1, and comprises a non-labeled nucleotide substrate other than the above non-labeled substrate.

11. The kit according to claim 10, wherein the reaction mixture further comprises a reverse transcriptase.