JP2003505038A - Methods for determining hybridization specificity and sensitivity of oligonucleotides - Google Patents

Abstract

  (57) [Summary] The present invention provides materials and methods that can be used to evaluate one or more different probes and to select probes that are optimized for sensitivity and specificity to a particular target. In a particularly preferred embodiment, the methods and compositions of the present invention can be used to evaluate polynucleotide probes having different nucleotide sequences. Thereby, the methods and compositions allow a user to select, for example, a polynucleotide probe having a particular nucleotide sequence that has been optimized for sensitivity and / or specificity to a particular target polynucleotide molecule. I do. In a particularly preferred embodiment, the methods and compositions can be used to evaluate multiple probes, such as on a microarray, simultaneously. The probes to be evaluated according to the method of the present invention are selected to select genomic polynucleotides (eg, genomic DNA) and genomic transcripts (eg, mRNA) or cDNA sequences derived therefrom, as well as special transcripts, single nucleotide polymorphisms, and the like. Can be used to detect a variety of molecules, including a variety of polynucleotides.

Description

Detailed Description of the Invention

1. Field of the Invention The field of the invention is that of genomic sequences, eg, by nucleic acid hybridization, on nucleic acid microarrays, genomic transcribed sequences (eg, mRNA obtained from cells and / or cDNA sequences derived therefrom). It relates to the field of detection and reporting of polynucleotide sequences including copy number and single nucleotide polymorphisms (SNPs). In particular, the present invention relates to methods for the identification and / or selection of polynucleotide sequences, particularly oligonucleotide sequences, which hybridization probes are sensitive and specific to a target polynucleotide sequence of particular interest (e.g. , On a nucleic acid microarray)
Can be used as

2. Background of the Invention Within the last decade, several techniques have made it possible to monitor the expression levels of multiple gene transcripts at any time (eg Schena et al., 1995, Sci.
ence 270: 467-470; Lockhart et al., 1996, Nature Biotechnology 14: 1675-1680;
Blanchard et al., 1996, Nature Biotechnology 14: 1649; Ashby et al., U.S. Pat.
569,588, issued October 29, 1996). For example, techniques for preparing microarrays of cDNA transcripts are known (eg DeRisi et al., 1996, Natur.
e Genetics 14: 457-460; Shalon et al., 1996, Genome Res. 6: 689-645; and Schen.
a et al., 1995, Proc. Natl. Acad. Sci. USA 93: 10539-11286).
. Alternatively, high density arrays containing thousands of oligonucleotides complementary to defined sequences at defined positions on the surface using photolithographic techniques for in situ synthesis have been described, for example, by Fodor et al., 1991. , Science 251: 767-773; Pease et al., 1994.
, Proc. Natl. Acad. Sci. USA 91: 5022-5026; Lockhart et al., 1996, Nature.
Biotechnology 14: 1675; US Pat. Nos. 5,578,832; 5,556,752; and 5
, 510,270. Methods for making arrays using inkjet technology for oligonucleotide synthesis are also known in the art (eg, Blanchard, International Patent Publication WO 98/41531, published September 24, 1998; Blanchard et al., 1996, Biosensor.
s and Bioelectronics 11: 687-690; Blanchard, 1998, Synthetic DNA Arrays.
in Genetic Engineering, Vol. 20, JK Setlow (ed.), Plenum Press, New York,
See pages 111-123).

Applications of this technology include, for example, the identification of genes that are upregulated or downregulated in various physiological conditions, particularly pathological conditions. Further examples of uses for transcript arrays include analysis of members of the signaling pathway and identification of targets for various drugs. For example, Friend and Hartwell,
International Patent Publication WO 98/38329 (Published September 3, 1998); Stoughton, US Patent Application No. 09
/ 099,722 (filed June 19, 1998); Stoughton and Friend, US patent application No. 09/074
, 983 (filed May 8, 1998); Friend and Stoughton, US Provisional Application No. 60 / 084,74
No. 2 (filed May 8, 1998), No. 60 / 090,004 (filed June 19, 1998), and No. 60/0
See No. 90,046 (filed June 19, 1998).

Oligonucleotide sequences are particularly useful as probes on microarrays and for other applications involving nucleic acid hybridization. Using any desired DNA sequence, techniques known in the art (e.g., Froehler et al., 1986, Nucleic Acid Res.
14: 5399-5407; McBride et al., 1983, Tetrahedron Lett. 24: 246-248), this oligonucleotide can be custom synthesized. Moreover, oligonucleotides whose thermodynamic properties (eg, free binding energies for complementary and / or partially complementary sequences) can be at least partially predicted are small enough. However, due to their small size, oligonucleotide probes often match non-unique genomic sequences, resulting in
It can hybridize to more than one polynucleotide sequence. For example, a particular oligonucleotide probe may not only hybridize to a particular mRNA transcript of interest in a sample, but may also be more abundant in other homologs, analogs, splice variants or samples. Even sequences that are less related to the transcript can hybridize. As a result of such "cross-hybridization," many oligonucleotide probes can give false-positive measurements that reflect a lack of specificity. Conversely, the oligonucleotide probe may also hybridize to the target polynucleotide sequence of interest weaker than expected (eg, expected hybridization binding energy). Such probes can yield false negative hybridization measurements that reflect a lack of sensitivity.

As a result of these limitations, empirically discriminate between signals resulting from a target polynucleotide sequence of interest (eg, a particular mRNA sequence of interest) and signals resulting from cross-hybridization with other polynucleotide sequences. In order to do so, current microarrays require multiple probe pairs that match the target sequence and intentionally mismatch. Currently, in situ synthesized microarray chips require more than 20 oligonucleotide probe pairs per reported gene or gene region (Lockhart et al., Supra). However, unless a large number of probes are used, such a match-mismatch scheme can only screen cross-hybridizations from less related sequences. In particular, the ability of such match-mismatch schemes to distinguish between true hybridization and cross-hybridization for closely related sequences (e.g., closely related homologs and splice variants) is typically Limited or extremely weak. Furthermore, the "reported density" of a microarray (ie, the number of genes detected per unit of surface area) is limited, for example, by the density at which the polynucleotide probes are stored as well as the number of polynucleotide probes required per gene. Therefore, the number of polynucleotide probes that can be stored on a microarray chip is limited by the technology used to fabricate the microarray. The above photolithography techniques for the fabrication of oligonucleotide microarrays with high spatial density probes require large capital investment due to the high cost of synthesis. In contrast, oligonucleotide microarrays manufactured using the inkjet technique described above are very cheap per chip design and per chip and can be manufactured more quickly. Therefore, such microarrays are generally preferred for the detection of gene transcripts in cells. However, the microarray chip manufactured by such an inkjet technique has a limited probe density, and the density is only a part of the probe density of the chip manufactured by the photolithography method. Therefore, microarrays presently known in the art use redundant probe numbers (e.g., 20) and need to have a limited probe density, so genes that can be efficiently detected on a single microarray chip. The number of transcripts is about 10,000 gene transcripts when using an expensive photolithographic array, and about 750 to 2,500 when using an inexpensive inkjet array.
Limited to individual gene transcripts.

Therefore, there is a need for a method of identifying specific oligonucleotide sequences that can be used as sensitive and specific probes for target polynucleotide sequences. In particular, it identifies a particular sequence that hybridizes to a particular sequence of interest, such as a particular gene or gene transcript, with little or no cross-hybridization to other polynucleotide sequences in the sample. A method that can do is needed. Also, less nucleic acid probe sequences are needed to detect individual genes of interest, and thus nucleic acids containing polynucleotide probe sequences for detecting more genes of interest than are currently available in the art microarrays. A method of designing the array is also needed.

Any discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION The present invention describes the binding properties of a first type of molecule (referred to herein as a probe molecule) to a second type of molecule (referred to herein as a target molecule). Provided are compositions and methods that can be used to evaluate. In particular, the methods and compositions of the present invention include
It can be used to assess the sensitivity and specificity of a probe for binding to a particular target.

As used herein, the term “probe sensitivity” refers to the absolute amount or level of a particular target that binds to the probe under particular binding conditions (ie, the number of molecules of the particular target).
Is understood to mean. The amount or level of a particular target that binds to a probe under particular binding conditions also refers herein to the amount or level of specific binding to a probe under particular binding conditions.

As used herein, the term “probe specificity” refers to the specific amount that binds a probe under the same binding conditions, relative to the amount or level of non-specific binding to the probe under specific binding conditions. It is understood to refer to the amount or level of target (ie the number of molecules of a particular target). As used herein, the term "non-specific binding" refers to the amount of molecules other than the specific target molecule that bind to the probe under specific binding conditions (i.e., the number of molecules that are not the specific target molecule). Is understood to mean.

The method of the invention compares the amount or number of molecules in a first sample that bind to a molecule of a probe with the amount or number of molecules in a second sample that bind to a molecule of the same probe. Including that. The first sample, referred to herein as the "specifically bound sample," preferably comprises a molecule of a particular target, which is generally the target of interest to the user. Preferably, the specific target molecule in the specific binding sample is substantially pure (e.g., at least 75% pure, preferably at least 90% pure, more preferably at least 95% pure, even more preferably 99%. pure).

A second sample, referred to herein as a “non-specific binding sample”, contains a plurality of different (ie, non-identical) targeting molecules other than the particular target of interest. Preferably, the plurality of different target molecules are of the same type and have about the same abundance as the molecules in the actual sample for which the probe is intended.

The present invention provides, at least in part, a discriminatively bound sample of a competing molecule that is of the same type and has about the same abundance as the competing molecule in the actual sample intended for the particular probe. It is based on the finding that by providing a meaningful measure of non-specific binding of a particular probe to a molecule.
Such bound samples can be readily obtained, for example, according to the methods of the invention described herein below, and can be readily compared to specific binding measurements obtained from specific binding samples. It can be used as a non-specific binding sample in the method of the invention for obtaining an actual measurement of possible non-specific binding. Indeed, the method of the present invention simultaneously measures specific and non-specific binding levels. Although the specific and non-specific binding samples need not be physically separated, it is possible to distinguish between the binding of molecules obtained from the specific binding sample and the binding of molecules obtained from the non-specific binding sample. It only needs to be identified from each other so that they can.
For example, in a preferred embodiment, specific and non-specifically bound samples are differentially labeled, eg, with fluorescent labels that fluoresce at different wavelengths.

The method of the present invention is particularly useful for evaluating a large number of different probes. For example, using the methods of the invention, the amount or number of molecules from a first sample (i.e., a specific binding sample) that binds to each of a plurality of different probes is bound to a plurality of different probes. Second sample (i.e. non-specifically bound sample)
Multiple different probes can be evaluated by comparison with the number or amount of molecules derived from. In a preferred embodiment, the method of the invention is used to evaluate a plurality of different probes in a probe array. The array comprises a solid phase (or, in certain embodiments, a semi-solid phase) support or surface on which a plurality of different probe molecules are immobilized. Most preferably, the array positions each different probe at a specific, known location on the support or surface such that the identity of a particular probe can be determined from its location on the support or surface. Accessible array, such as a locally accessible array.

The probes and target molecules that can be evaluated using the compositions and methods of the present invention can be of any type, but are preferably of the type or class capable of specifically binding to each other. For example, in certain embodiments, the probe may be a molecule of a particular antibody (preferably a monoclonal antibody) and the target molecule may be a molecule to which an antibody such as a protein can specifically bind. However, the compositions and methods of the present invention differ to test the sensitivity and specificity of a polynucleotide probe when hybridizing to a particular target polynucleotide (i.e., a polynucleotide molecule having a particular nucleotide sequence). It is particularly useful for assessing the hybridization properties of polynucleotide probes. Therefore,
In a preferred embodiment of the invention, the probe and target molecule are both polynucleotide molecules.

In such preferred embodiments, the sensitivity of the probe is the absolute amount of a particular target polynucleotide that hybridizes with the probe under particular hybridization conditions (ie, the number of polynucleotide molecules having a particular nucleotide sequence). ) Is understood. Also, the amount of a specific target polynucleotide that hybridizes with a probe under specific hybridization conditions is referred to herein as the amount of specific hybridization with a probe under specific hybridization conditions.

The term “probe specificity” as used in a preferred embodiment of the present invention, refers to the amount of cross-hybridization to a probe under specific hybridization conditions, or to the same conditions. It is understood below to refer to the amount of a particular target polynucleotide that hybridizes to a probe (ie the number of polynucleotide molecules having a particular nucleotide sequence).

“Cross-hybridization” or “non-specific hybridization” as the term is used herein refers to a polynucleotide other than a specific target polynucleotide that hybridizes with a probe under specific hybridization conditions. It is understood to refer to an amount (ie, the number of polynucleotide molecules having a nucleotide sequence that differs from the nucleotide sequence of a particular target polynucleotide).

In a particularly preferred embodiment, the method of the invention comprises determining the number or amount of polynucleotide molecules from the first sample that hybridize with the molecules of the polynucleotide probe to the molecules of the polynucleotide probe that hybridize. Comparing the number or amount of polynucleotide molecules from two samples. The first sample is a "specific hybridization sample" containing molecules of a particular target polynucleotide (ie, polynucleotide molecules having a particular sequence). The polynucleotide sequence may be, for example, the sequence of a particular gene or gene transcript of a cell or organism. The second sample is a "non-specific hybridization sample" that contains a plurality of different (ie, non-identical) polynucleotide molecules, each different polynucleotide molecule having a different nucleotide sequence. In particular, the second sample or non-specific hybridization sample should include a polynucleotide molecule having a nucleotide sequence that differs from the nucleotide sequence of the particular target polynucleotide in the first sample or specific hybridization sample. . For example, in an embodiment in which the sequence of the particular target polynucleotide in the first sample or the specific hybridization sample is the sequence of a particular gene or gene transcript of a cell or organism,
The nucleotide sequence of the polynucleotide molecule in the non-specific hybridization sample of 2 preferably comprises sequences indicative of other genes or gene transcripts of the cell or organism.

For example, the invention provides that the first sample (ie, the specific hybridization sample) is substantially pure of molecules having a particular target nucleotide sequence (ie, at least 75% pure, preferably at least 90%). % Pure, more preferably at least 95% or 99% pure) and the second sample (i.e. non-specific hybridization sample) comprises a plurality of different polynucleotide molecules, each different polynucleotide molecule. Provides a first preferred embodiment which is a second sample having a different nucleotide sequence. In a particularly preferred aspect of this first preferred embodiment, the target polynucleotide molecule is a gene or gene transcript (e.g., mRNA or cDNA molecule) of a cell or organism,
A non-specific hybridization sample is a "deletion mutant" of the cell or organism (
That is, a polynucleotide sample obtained from a cell or organism variant or strain that is not expressed because the gene or gene transcript corresponding to the target polynucleotide is absent or silent.

The invention also provides that the first sample (ie, the specific hybridization sample) is a substantially pure sample of molecules having a particular target nucleotide sequence, and the second sample (ie, the non-specific sample). Hybridization sample)
Also comprises a plurality of different polynucleotide molecules, wherein each different polynucleotide molecule in the second sample has a different nucleotide sequence. In particular, in a second preferred embodiment of the present invention, the polynucleotide molecule in the non-specific hybridization sample comprises a molecule of the target polynucleotide sequence. In a particularly preferred aspect of this second preferred embodiment, the target polynucleotide corresponds to a gene or gene transcript of a cell or organism and the non-specific hybridization sample is a gene or gene transcript corresponding to the target polynucleotide. "Wild type" that expresses at a normal level or amount
Is a polynucleotide sample obtained from the cells or organisms of.

The invention further comprises that the first sample (ie the specific hybridization sample) comprises molecules having a particular target polynucleotide sequence as well as molecules having other target polynucleotide sequences and the second sample (ie A non-specific hybridization sample) comprises a plurality of different non-target polynucleotide molecules. Therefore, in a third preferred embodiment of the present invention, the specific hybridization sample is the same as the non-specific hybridization sample described above for the second preferred embodiment of the present invention. Is the same as the non-specific hybridization sample described above for the first preferred embodiment of the present invention.

The invention further provides a fourth preferred embodiment, wherein both the first and second samples (ie, specific and non-specific hybridization samples) comprise a plurality of different polynucleotide molecules, such as molecules of a target polynucleotide. Embodiments are provided. However, in a fourth preferred embodiment of the invention, the amount or level of molecules of the target polynucleotide in the first specific hybridization sample is such that the amount of target polynucleotide in the second non-specific hybridization sample is The amount or level of the molecule is substantially different.

The probe used in the present invention may comprise any type of polynucleotide, but in a preferred embodiment the probe is an oligonucleotide sequence,
That is, it comprises a polynucleotide sequence of about 4 to about 200 bases in length, most preferably about
It comprises a polynucleotide sequence of 15 to about 150 bases in length. In one embodiment, about 40
Use shorter oligonucleotide sequences that are less than base length, more preferably about
Oligonucleotide sequences that are 15 to about 30 bases long are used. However, in a preferred embodiment of the present invention, a longer oligonucleotide probe that is about 40 to about 80 bases in length is used, and an oligonucleotide sequence that is about 50 to about 70 bases in length (e.g.,
It is particularly preferable to use an oligonucleotide sequence having a length of about 50 to 60 bases.

The compositions and methods of the invention can also be used to evaluate different binding conditions for a probe or multiple different probes. For example, the compositions and methods of the present invention can be used to assess hybridization conditions for one polynucleotide probe or multiple different polynucleotide probes. In particular, the amount of binding of a molecule derived from a first binding sample (i.e., a specific binding sample) under specific binding conditions to one or more different probes is determined by the second binding under the same binding conditions. The amount of binding of molecules from the sample (ie, non-specifically bound sample) to one or more different probes can be compared. The sensitivity and specificity of the probe under particular binding conditions can then be readily determined. Thus, by performing the above comparison under different binding conditions (e.g., different hybridization conditions for polynucleotides), for example, to determine the binding conditions that optimize the sensitivity and specificity of the probe for a particular target. Thus, the optimum binding condition can be easily confirmed.

Therefore, the present invention provides, in the first embodiment, a method for evaluating a probe, which comprises comparing the amount of the first sample bound to the probe with the amount of the second sample bound to the probe. To do. The first sample contains a specific target molecule,
The second sample contains a plurality of different target molecules. In one preferred aspect of this first embodiment, the first sample is substantially pure (e.g., at least 75%, at least 90%, at least 95%, or at least 99% pure) of a particular target molecule.
It is a sample. In another aspect of this first embodiment, each different target in the plurality of different targets of the second sample is different from the particular target of the first sample. In yet another preferred aspect of this embodiment, the method also preferably comprises binding of a specific target molecule in the first sample to the probe and a plurality of target molecules in the second sample to the probe. The specificity of the probe is determined from the ratio with the amount bound. The invention further provides the aspect of the first embodiment above, wherein the molecules of the first and / or second sample are detectably labeled, eg with a fluorescent molecule. In a particularly preferred embodiment, a particular target molecule in a first sample is detectably labeled with a first label and a plurality of target molecules in a second sample is detectably labeled with a second different label. (Eg, the first and second fluorescent molecules). In a preferred aspect of this embodiment, the probe is attached to the surface of the support. In another preferred embodiment, the probe is one of a plurality of probes, the plurality of probes comprises an array of probes comprising a support having at least one surface with each probe bound to the surface. Including.

Also provided is an aspect of this first embodiment, wherein the probe is a polynucleotide probe having a particular nucleotide sequence. In various other embodiments provided by the present invention, the particular target molecule in the first sample is a polynucleotide molecule having a nucleotide sequence. In a particularly preferred embodiment, the polynucleotide probe is attached to the surface of the support. In another preferred embodiment, the polynucleotide probe is a plurality of polynucleotide probes.
One, and preferably the plurality of polynucleotide probes comprises an array of polynucleotide probes comprising a support having at least one surface with each polynucleotide probe bound to the surface.

The present invention also provides, in another embodiment, a method for evaluating a polynucleotide probe having a specific nucleotide sequence, said method comprising the amount of hybridization of the first sample to the polynucleotide probe, Comparing the amount of hybridization of the second sample to the nucleotide probe, where the first sample comprises molecules of the target polynucleotide having the target nucleotide sequence and the second sample is different from each other. The polynucleotide molecule comprises a plurality of different polynucleotide molecules having different nucleotide sequences. In one preferred aspect of this other embodiment, the first sample is a substantially pure (eg, at least 75%, 90%, 95% or 99% pure) sample of the molecule of the target polynucleotide. In one aspect, each different polynucleotide molecule in the second sample has a nucleotide sequence that differs from the target nucleotide sequence. In another aspect, the plurality of different polynucleotide molecules in the second sample are different from (a) a polynucleotide molecule having a nucleotide sequence that is identical to the target nucleotide sequence, and (b) a target nucleotide sequence. It comprises a plurality of different polynucleotide molecules each having a different nucleotide sequence. In another embodiment, the first sample further comprises a polynucleotide molecule having a nucleotide sequence that differs from the target nucleotide sequence. In one aspect, each different polynucleotide molecule in the second sample has a nucleotide sequence that differs from the target nucleotide sequence. In another embodiment, the second sample is (a) a polynucleotide molecule having a nucleotide sequence that is identical to the target nucleotide sequence,
And (b) comprising a plurality of different polynucleotide molecules, each different polynucleotide molecule having a different nucleotide sequence that is different from the target nucleotide sequence, wherein in the first sample having the target nucleotide sequence. The amount of polynucleotide molecule is substantially different (eg, at least 2, at least 4, at least 8, at least 20, or at least 100-fold different) from the amount of polynucleotide molecule in the second sample having the target nucleotide sequence.

The present invention also provides, in yet another embodiment, a method for evaluating a plurality of polynucleotide probes, wherein each polynucleotide probe in the plurality of polynucleotide probes has a specific nucleotide sequence. The method determines the amount of hybridization of a first sample to each polynucleotide probe in a plurality of polynucleotide probes and the amount of hybridization of a second sample to each polynucleotide probe in a plurality of polynucleotide probes. Comparing, wherein the first sample comprises molecules of a target polynucleotide having a target nucleotide sequence and the second sample comprises a plurality of different polynucleotides, each different polynucleotide molecule having a different nucleotide sequence. Contains nucleotide molecules.

The present invention further provides, in yet another embodiment, a method for assessing hybridization conditions for one or more polynucleotide probes, each probe having a particular polynucleotide sequence. The method comprises the amount of hybridization of a first sample to each of the one or more polynucleotide probes under specific hybridization conditions and the amount of hybridization of the one or more polynucleotide probes under the same specific hybridization conditions. Comparing the amount of hybridization of a second sample for each. The first sample preferably contains molecules of the target polynucleotide having the target nucleotide sequence. The second sample comprises a plurality of different polynucleotide molecules, preferably each different polynucleotide molecule having a different nucleotide sequence.

4. Brief Description of the Drawings (see below for description of the drawings) 5. Detailed Description This section provides a detailed description of the invention and its application. This description is given in more detail and more specifically with respect to the general method of the present invention by means of some representative descriptions. These examples are non-limiting and related modifications that will be apparent to those skilled in the art are encompassed by the claims.

In particular, the invention relates to methods and compositions that can be used to assess the properties of different probe molecules, particularly the sensitivity and specificity of probe binding to a particular target. In a particularly preferred embodiment both the probe molecule and the target molecule are polynucleotides. Accordingly, the methods and compositions of the present invention are primarily described below for these embodiments (ie, for probe and targets that are polynucleotide molecules).

However, one of ordinary skill in the art can readily discern other embodiments in which the methods and compositions of the present invention can be utilized to evaluate different types of probe and target molecules. . In fact, it is preferred that the probe and target molecules are of the type and kind that are capable of specifically binding to each other, although the invention is equally applicable to any type of probe and target molecule. . For example, if the target molecule can be any type of molecule to which an antibody can specifically bind, it will be appreciated by those skilled in the art that in certain alternative embodiments, the probe of the invention may include an antibody (preferably a monoclonal antibody). Can be easily recognized. For example, the target molecule of the present invention may also be a protein or peptide molecule. In addition, one of ordinary skill in the art will recognize other alternative embodiments and can construct and use such alternative embodiments without undue experimentation. It is therefore understood that such alternative embodiments are also within the scope of the claims.

First, Section 5.1 provides an introduction to the present invention and, in particular, presents and defines certain concepts of the invention, including the concepts of probes and targets. Next, Section 5.2 details specific methods and compositions of the present invention. In particular, this summary describes methods for selecting and preparing candidate probes for evaluation (Section 5.2.1).
, A description of hybridization samples for use in the method of the invention,
Section 2.2) and a description of how hybridization levels can be measured for each of the two samples (Section 5.2.3). Also, Section 5.2.4 provides a method by which such hybridization data can be analyzed, for example, assessing one or more probes and determining the sensitivity and / or specificity of one or more probes. It also describes the methods that can be determined.

The methods and compositions of the present invention have numerous useful applications, such as, for example, probe selection and preparation for microarrays. For some of these uses,
See Section 5.3 below. Furthermore, finally, representative examples of the methods and compositions of the present invention are provided in Section 6 below. In particular, this example shows that the yeast Saccharomyces
One specific, but non-limiting embodiment of the present invention is described that uses the methods and compositions of the invention to evaluate candidate probes for the Saccaromyces cerevisiae gene YER019W.

5.1. Introduction The present invention provides the property of binding a given type of molecule (referred to herein as a probe molecule) to a second type of molecule (referred to herein as a target molecule). Methods and compositions that can be used for evaluation are provided. The term probe or probe molecule as used herein is understood to be any molecule that can be used to detect another molecule. Similarly, the term target or target molecule as used herein is understood to be any molecule that can be detected by using a probe.

In general, the target molecule is detected by detecting binding of the target molecule to the probe molecule. For example, in a preferred embodiment, the probe molecule is a solid (
Or, in some embodiments, semi-solid) immobilized on a support or surface. The sample is then bound to a solid support or surface under conditions that allow the target molecule to be detected by the probe molecule to bind to the probe molecule. . Then, the molecule not bound to the probe molecule is removed, but the support or surface is washed under the condition that the probe molecule and the target molecule are bound. The molecules in the sample are preferably detectably labeled with a fluorescent label or dye. Thus, binding of the target molecule to the probe molecule can be detected, for example, by a detectable label.

[0038] Preferably, the specific probe specifically detects a specific target. For example, in a preferred embodiment, the probe and target molecules are polynucleotide molecules, and preferably the particular probe molecule is a particular polynucleotide sequence (e.g.,
A polynucleotide molecule having a nucleotide sequence that is complementary to the nucleotide sequence of the probe molecule) is detected. Furthermore, the polynucleotide sequence to be detected is
Often there are hundreds, thousands, or hundreds of thousands of sequences in a particular sample. Therefore, it is generally preferred that the probe specifically binds to its target, especially for the probe to detect a particular target. In particular, it is generally preferred that the specific binding of a particular target to the probe be maximal, while the non-specific binding of the molecule to the probe be minimised.

As mentioned above, the probe and target are preferably molecules or molecules of the type or species that specifically bind to each other, but may include molecules of any type or species. For example, antibodies are useful probes for detecting molecules such as proteins and peptides to which the antibody specifically binds.

In a particularly preferred embodiment of the invention, the probe and target are both polynucleotide molecules. In particular, polynucleotide molecules (generally,
The molecules are characterized by their nucleotide sequences) and can bind or hybridize to other polynucleotide molecules by forming non-covalent Watson-Crick base pairs. Therefore, a target polynucleotide molecule having a particular nucleotide sequence is readily detectable by a polynucleotide probe molecule having a complementary sequence.

The term “target” polynucleotide, as used herein, refers to a molecule of a particular polynucleotide sequence of interest. Representative target polynucleotides that can be analyzed by the methods and compositions of the invention include genomic DNA molecules, cDNA molecules, and oligonucleotides, ESTs (evpressed sequence tags), STSs (sequencs).
Examples thereof include DNA molecules such as fragments thereof, such as e tag site), but are not limited thereto. Target polynucleotides that can be analyzed by the methods and compositions of the invention also include messenger RNA (mRNA) molecules, ribosomal RNA.
(rRNA) molecule, cRNA (i.e., RNA prepared from a cDNA molecule transcribed in vivo)
Molecules), and RNA molecules such as fragments thereof, but are not limited thereto.

The target polynucleotide may be from any source. For example, the target polynucleotide molecule can be a naturally occurring nucleic acid molecule such as a genomic or extragenomic DNA molecule isolated from a cell or organism, or an RNA molecule such as an mRNA molecule isolated from a cell or organism. Alternatively, the polynucleotide molecule may be synthesized, for example, a nucleic acid molecule enzymatically synthesized in vivo or in vitro such as a cDNA molecule, or a polynucleotide molecule synthesized by PCR, in vitro.
RNA molecules and the like synthesized by transcription are included. The sample of target polynucleotide may include, for example, molecules of DNA, RNA or a copolymer of DNA and RNA. In a preferred embodiment, the target polynucleotide of the invention comprises a specific gene or a specific gene transcript (e.g., a specific mRNA sequence expressed in a cell, or such mRNA).
Corresponding to a specific cDNA sequence derived from an RNA sequence). However, many embodiments (
In particular, in embodiments where the polynucleotide molecule is derived from a mammalian cell), the target polynucleotide may correspond to a particular fragment of the gene transcript. For example,
The target polynucleotide may correspond to different exons of the same gene, so that, for example, different splice variants of that gene can be detected and / or analyzed.

Preferably, the polynucleotide analyzed by the compositions and methods of the present invention is of cell or organism origin. In particular, the target polynucleotide is a particular gene or particular gene transcript (e.g., mRNA derived therefrom) of a cell or organism.
Sequence or cDNA sequence) and is identical. Therefore, the probe evaluated using the methods and compositions of the present invention is preferably, for example, of cell or organism origin, and therefore the specific gene and The purpose is to detect specific gene transcripts.

Particularly preferred cells and organisms are those that can be manipulated, for example by in vitro means by homologous recombination and / or conventional methods of sexual genetics. More specifically, preferred cells or organisms are cells that are readily available for a particular defective strain or mutant (e.g., a strain in which one or more particular genes or subjects are deleted). Or, a living thing is included. Such cells and organisms include bacterial cells and organisms such as E. coli,
And yeast cells and organisms, such as Saccharomyces cerevisiae, are examples. In addition, other cells and organisms from which specific deletion strains or mutants can be easily obtained without undue experimentation will be apparent to those skilled in the art.

However, the methods and compositions of the present invention include, for example, plant cells as well as cells of higher organism origin, such as animal cells, including mammalian cells such as cells of mouse, rat or human organism origin. It is understood that it can be used to evaluate probes for polynucleotides of any cell or organism, including The methods and compositions of the invention can be used to evaluate probes for polynucleotides of biological origin, even if a particular deletion strain or mutant is not available.

Briefly, although the present disclosure often refers to a single cell,
(E.g., "isolating RNA from cells"), any particular step of the invention is performed, for example, using a number of genetically and transcriptionally identical cells from a cultured cell line. It is understood that there are more cases. Also, such similar cells are referred to herein as "
Cell type ". Cells of a particular cell type may be derived from natural unicellular organisms such as yeast or bacteria, or from multicellular higher organisms. However, it is also understood that the methods of the invention can also be practiced with samples (eg, mRNA samples) extracted from a large number of cells present in a tissue sample, eg, from a living organism, such as a patient. In general, the cells in such samples are genetically identical, but they generally contain different cell types of the organism that express at least some different genes. However, in other examples, the cells may be cells such as cancer cells (which contain one or more genetic mutations and are therefore not genetically identical).

In a preferred embodiment, the target polynucleotide to be analyzed is prepared in vitro from nucleic acid extracted from cells. For example, in certain embodiments, RNA is extracted from cells (eg, total cellular RNA) and messenger RNA is purified from the extracted total RNA. The cDNA is then synthesized from the purified mRNA using, for example, oligo-dT or random primers. Preferably, the target polynucleotides represent the original nucleic acid population of the cell, short polynucleotide molecules and / or fragmented polynucleotide molecules.

The target polynucleotides analyzed by the methods and compositions of the present invention are preferably detectably labeled. For example, cDNA can be labeled with a second labeled cD either directly using a nucleotide analog or the like, or using the first strand as a template.
Labeling can be done indirectly by creating NA chains. Or double-stranded
cDNA can be transcribed into cRNA and labeled.

Preferably, the detectable label is a fluorescent label, eg by incorporation of nucleotide analogues. Other labels suitable for use in the present invention include biotin, immunobiotin, antigens, cofactors, dinitrophenol, lipoic acid, olefin compounds,
Included are, but are not limited to, detectable polypeptides, electron-enhancing molecules, enzymes capable of producing a detectable signal by reaction on a substrate, and radioisotopes. Preferred radioisotopes include ³² P, ³⁵ S, ¹⁴ C, ¹⁵ N and ¹²⁵ I, which is an example. Suitable fluorescent molecules for the present invention include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, Texas Red, 5'-carboxyfluorescein (``
FMA ''), 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxy-fluorescein (`` JO
E "), N, N, N ', N'-tetramethyl-6-carboxy-rhodamine (" TAMRA "), 6'-
Carboxy-X-rhodamine (“ROX”), HEX, TET, IRD40 and IRD41. Furthermore, suitable fluorescein molecules for the present invention include Cy2, Cy3, Cy3.5, Cy5.
Cyamine, including, but not limited to, Cy5.5, Cy7 and FluorX
Dye, BODIPY-FL, BODIPY-TR, BODIPY-TMR, BODIPY-630 / 650 and BODIPY-650 / 6
BODIPY dyes including but not limited to 70, ALEXA-488, ALEXA
-ALEXA dyes, including but not limited to -532, ALEXA-546, ALEXA-568 and ALEXA-594, as well as other fluorescein dyes known to those skilled in the art. Suitable electron enhancing indicator molecules for the present invention include aferritin (aferri
tin), hemocyanin and colloidal gold, but not limited thereto. Alternatively, in less preferred embodiments, the labeled polynucleotide can be labeled by specifically binding the first group to the polynucleotide. The second group, which is covalently attached to the indicator molecule and has an affinity for the first group, can be used to indirectly detect the target polynucleotide. In such an embodiment,
Compounds suitable for use as the first group include, but are not limited to, biotin and immunobiotin.

The target polynucleotide that is analyzed (eg, detected) by the methods and compositions of the present invention is one that is under conditions such that a polynucleotide molecule having a sequence complementary to the probe hybridizes to the probe. It binds to a probe or to multiple probes. As used herein, a "probe" means a polynucleotide molecule of a particular sequence, a target polynucleotide molecule having a particular sequence (generally a sequence complementary to the probe sequence) is the target polynucleotide molecule for the probe. Can be hybridized so that the hybridization can be detected. The polynucleotide sequence of the probe can be, for example, a DNA sequence, an RNA sequence, or a sequence of a DNA and RNA copolymer. For example, the probe polynucleotide sequence may be genomic DNA, cDNA, mRNA or cRN extracted from a cell.
It may be the complete sequence or a partial sequence of the A sequence. Furthermore, the polynucleotide sequence of the probe can also be synthesized, for example, by oligonucleotide synthesis techniques known to those skilled in the art. The probe sequence can also be synthesized enzymatically in vivo, enzymatically in vitro (eg, by PCR), or non-enzymatically in vitro.

Preferably, the probe used in the method of the invention is a polynucleotide sequence that is not hybridized or bound to the probe without removing the probe and all polynucleotides bound or hybridized thereto. Are immobilized on a solid support or surface so that they can be washed or removed. In certain embodiments, the probes comprise separate polynucleotide arrays bound to a solid (or semi-solid) support or surface such as a glass surface. More preferably, the array is an addressable array, where each different probe is located at a particular known location on the support or surface, so that the identification of the particular probe is determined by the support or It can be determined from its position on the surface.

The probes used in the present invention include polynucleotides of all types, but in a preferred embodiment, the probes include oligonucleotide sequences (for example, from about 4 to about 200 bases in length, and Preferably, a polynucleotide sequence having a base length of about 15 to about 150) is included. In certain embodiments, shorter oligonucleotide sequences that are about 4 to about 40 bases long, and more preferably about 15 to about 30 bases long are used. However, in a more preferred embodiment of the present invention, a longer oligonucleotide probe of about 40 to about 80 bases in length is used, and particularly preferred is an oligonucleotide sequence of about 50 to about 70 bases in length (e.g., about 60 bases). (Long oligonucleotide sequence) is used.

The present invention is based, at least in part, on the finding that non-specific hybridization to a particular probe can be assayed by providing a distinguishable hybridization sample of competing molecules. In particular, non-specific hybridization samples are readily available, which include polynucleotide sequences other than the specified sequences that are adapted to specifically hybridize to the probe. Rather, the polynucleotide sequence of such a non-specific hybridization sample competes with the same type of polynucleotide and has the same abundance when the probe competes with the polynucleotide in the experimental sample of interest.

5.2. Overview of the Method of the Invention A flowchart illustrating an exemplary, non-limiting embodiment of the invention is shown in FIG. This particular embodiment evaluates the hybridization properties of a plurality of different polynucleotide probes on a microarray, and more particularly, it is used to sensitize each of the different probes to hybridize to a particular target polynucleotide. And / or specificity can be assessed.

In the particular embodiment shown in FIG. 1, a microarray or “chip” of polynucleotide probes is designed and produced by first selecting a plurality of different oligonucleotide sequences for evaluation by the method of the invention ( 101), followed by synthesis of a microarray of selected oligonucleotide sequences (102), for example by inkjet synthesis techniques.

Two differently labeled hybridization samples: a specific hybridization sample (103) and a non-specific hybridization sample (104) are also prepared. For example, in the particular embodiment shown in Figure 1, the specific hybridization sample (103) is a purified labeled polynucleotide labeled with a green fluorescent label (eg, a fluorescent dye such as Cy3 that fluoresces green when stimulated). A "gene-specific" sample containing (eg, a polynucleotide molecule of the purified gene of interest). An exemplary non-specific hybridization sample (104) has a red fluorescent label (eg, Cy that fluoresces red when stimulated).
Derived from a "deletion strain" of cells or organisms (ie, a fluorescent dye such as 5) (ie,
Sample of a polynucleotide from a cell or strain of organism that does not express the target polynucleotide). Thus, a non-specific hybridization sample is a polynucleotide sample in which the target polynucleotide has been removed or deleted.

Both specific and non-specific hybridization samples are hybridized (105) with the probe, preferably simultaneously, and the intensity of each label is measured. The signal intensity and / or ratio is then analyzed (106) and used to assess the hybridization properties of the various probes.

In the following, with respect to general aspects of the invention, the steps shown in FIG. 1 will be described in detail by means of special exemplary embodiments. In particular, Section 5.2.1, below, describes certain exemplary methods by which candidate probes may be selected for evaluation by the methods of the invention. In a particularly preferred embodiment of the invention, the candidate probes comprise a microarray of probes. Therefore, Section 5.2.1 also operably describes microarrays and methods for preparing candidate probes for microarrays.

Section 5.2.2 operably describes both specific and non-specific hybridization samples for use in the invention to evaluate candidate probes. This description includes a description of particularly preferred embodiments of both specific and non-specific hybridization samples that can be used in the present invention. Exemplary methods and compositions for labeling such samples, including the preferred identification labeling method of the present invention, are also described in Section 5.2.2. Section 5.2.3 describes a method for measuring the hybridization of two samples (ie specific and non-specific hybridization samples) to a candidate probe, including a description of suitable hybridization conditions. Finally, Section 5.3 describes methods by which the hybridization data thus obtained can be analyzed, for example for assessing the sensitivity and specificity of individual probes for target polynucleotides.

5.2.1 Selection of Candidate Probes In the exemplary method shown in FIG. 1, one or more polynucleotide probes are provided and selected for analysis according to the methods of the invention. In particular, the polynucleotide probe analyzed according to the method of the present invention comprises a nucleotide sequence capable of hybridizing to the target polynucleotide molecule under suitable hybridization conditions. Generally, the target polynucleotide molecule that hybridizes to the probe is
It has at least one nucleotide sequence that is complementary to the nucleotide sequence of the probe and thus can hybridize to the probe by forming non-covalent Watson-Crick base pairs with the nucleotides of the probe sequence. Accordingly, polynucleotide probes can be selected or provided by selecting or providing polynucleotide probes having different nucleotide sequences. Since the preferred target polynucleotide molecule of the present invention is a polynucleotide molecule corresponding to a gene or gene transcript of a cell or organism, the selected or provided polynucleotide probe is preferably a nucleotide sequence of the gene or gene transcript of interest. Has a nucleotide sequence complementary to at least a part of

In a preferred embodiment, the polynucleotide probe selected or provided for analysis is an oligonucleotide probe, ie a probe comprising an oligonucleotide sequence. The oligonucleotide sequence is preferably about 4 to about 20.
A short sequence of polynucleotides that is between 0 bases (ie nucleotides) long, more preferably between about 15 and about 150 bases long. In one embodiment,
Shorter oligonucleotide sequences of less than about 40 bases long, preferably between about 15 and 30 bases long are used. However, in a preferred embodiment of the invention, a longer oligonucleotide sequence of between about 40 and about 80 bases in length is used,
Oligonucleotide sequences of between 0 and about 70 bases in length are preferred, from about 50 to about 6
More preferred are oligonucleotide sequences between 0 bases in length.

The compositions and methods of the present invention can be used to assess the hybridization properties of any probe or probes that generally include polynucleotide sequences immobilized on a solid support or surface. For example, as mentioned above, the probe is DNA
It may include sequences, RNA sequences, or copolymer sequences of DNA and RNA. The polynucleotide sequence of the probe may also include DNA and / or RNA analogs or combinations thereof. For example, the polynucleotide sequence of the probe can be the full or partial sequence of genomic DNA, cDNA, mRNA or cRNA sequence extracted from the cell. The probe polynucleotide sequence can also be a synthesized nucleotide sequence, such as a synthetic oligonucleotide sequence. The probe sequence is enzymatically and in vitro in vivo.
It can be synthesized enzymatically in vitro (eg by PCR) or non-enzymatically in vitro.

The one or more probes used in the methods and compositions of the present invention are immobilized on a solid support, which may preferably be porous or non-porous. For example, the probe can be a nitrocellulose or nylon membrane or a polynucleotide sequence attached to a filter. Such hybridization probes are well known in the art (eg Sambrook et al. Eds., 1989, Molecular Cloning: A Laborat.
ory Manual, 2nd edition, Vol. 1-3, Cold Spring Harbor Laboratory, New York). Alternatively, the solid support or surface may be a glass or plastic surface, or it may be a semi-solid support such as a gel.

Microarray General In a particularly preferred embodiment, a population of DNA or DNA mimics, or alternatively RNs.
The hybridization level is measured for a microarray of probes consisting of a solid phase having a population of polynucleotides such as A or a population of RNA mimics immobilized on the surface. The solid phase may be non-porous or it may be a porous material such as gel. Microarrays can be used to analyze the transcriptional status of cells, eg, the transcriptional status of cells exposed to graded levels of a drug of interest, or graded perturbations to a biological pathway of interest. Microarrays are particularly useful in the methods of the present invention in that they can be used to simultaneously screen a plurality of different probes, eg, to assess the sensitivity and specificity of each probe for a particular target polynucleotide.

In a preferred embodiment, the microarray comprises a support or surface having, for example, an ordered array of binding (eg, hybridization) sites for a plurality of different probes. Microarrays can be made in many ways, some of which are described below. However, no matter how they are made, microarrays share certain properties. That is, the array is reproducible, multiple copies of a given array can be made, and can be easily compared to each other. Preferably, microarrays are made from materials that are stable under binding (eg, nucleic acid hybridization) conditions. Microarrays are preferably small, eg between about 5 cm ² and 25 cm ² , preferably about 12 to 1
It is between 3 cm ² . However, larger arrays can be envisioned,
It may be preferred, for example for evaluating a large number of different probes simultaneously.

Preferably, a given binding site, or unique set of binding sites, in a microarray is associated with a single gene or gene transcript from a cell or organism (eg, a particular mRNA or a particular cDNA derived therefrom). Will specifically bind (eg hybridize). However, as mentioned above, other related or similar sequences will generally cross-hybridize to a given binding site.

Microarrays for use in the methods and compositions of the invention include one or more test tubes, each having a polynucleotide sequence complementary to a subsequence of RNA or DNA to be detected. . Each probe preferably has a different nucleic acid sequence and the position of each probe on the solid surface of the array is preferably known. In fact, the microarray is preferably an addressable array,
More preferably a location addressable array. More specifically, each probe of the array is preferably at a known, predetermined position on the solid support,
The identity (ie sequence) of each probe is such that its position on the array (ie on the support or surface) can be determined.

[0068]   Preferably, the density of the probes on the microarray is 1 cm^TwoAbout 100 per
Of different (ie non-identical) probes or more. More preferably
, The microarray used in the method of the present invention is 1 cm^TwoAt least 5
50 probes, 1 cm^TwoAt least 1,000 probes per cm, 1 cm ^Two At least 1,500 probes per cm, or 1 cm^TwoAt least
It has 2,000 probes. In a particularly preferred embodiment,
Rays are preferably of at least about 2,500 different probes per cm2.
A high density array having a density. Therefore, the micro array used in the present invention is
A is preferably at least 2,500, at least 5,000, less
10,000 pieces, at least 15,000 pieces, at least 20,000 pieces,
At least 25,000, at least 50,000, or at least 55
Includes 1,000 different (ie non-identical) probes.

In one embodiment, a microarray is an array (ie, matrix) where each position represents a distinct binding site for a gene-encoded product (ie, mRNA or cDNA derived therefrom). For example, the binding site can be DNA or a DNA analog to which a particular RNA can specifically hybridize. The DNA or DNA analog can be, for example, a synthetic oligomer, a full length cDNA, a cDNA shorter than full length, or a gene fragment.

Preferably, the microarrays used in the present invention have binding sites (ie, probes) for one or more genes associated with the action of the drug of interest or in the biological pathway of interest. "Gene" is messenger RN
A is identified as an open reading frame (ORF) that encodes a sequence of preferably at least 50, 75, or 99 amino acid residues that is transcribed in a cell or cells of an organism or multicellular organism. The number of genes in a genome can be evaluated from the number of mRNAs expressed by the cell or organism or by extrapolation from well characterized genomic parts of the genome. When the genome of the organism of interest is sequenced, the number of ORFs can be determined and analysis of the DNA sequence can identify the mRNA coding region. For example, the genome of Saccharomyces cerevisiae has been fully sequenced, with approximately 6275 ORs longer than 99 amino acid residues in length.
It has been reported to have a sequence encoding F. Analysis of these ORFs indicates that there are 5,885 ORFs that would encode protein products (Go
ffeau et al., 1996, Science 274: 546-567). In contrast, the human genome is estimated to contain approximately 10 ⁵ genes.

Preparation of Probes for Microarrays As mentioned above, a "probe" to which a particular target polynucleotide molecule specifically hybridizes in accordance with the present invention is a polynucleotide sequence which is complementary to the target polynucleotide. . In one embodiment, the microarray probes are
It contains sequences longer than 500 nucleotide bases in length corresponding to a gene or gene fragment. For example, such probes can include DNA or DNA "mimetics" (eg, derivatives and analogs) corresponding to at least a portion of one or more genes in the genome of an organism. In other embodiments, such probes are complementary RNA or R
It is a simulated NA.

DNA mimetics are polymers consisting of subunits that are capable of specific Watson-Crick-like hybridization with DNA or specific hybridization with RNA. For example, DNA mimetics can include nucleic acids with modified base moieties, sugar moieties, or phosphate backbones. For example, but not limited to, one special DNA mimetic is phosphorothioate.

Such DNA sequences may be, for example, genomic DNA, mRNA (for example from RT-PCR),
Alternatively, it may be obtained, for example, by polymerase chain reaction (PCR) amplification of a gene fragment from the cloned sequence. PCR primers are preferably selected based on the known sequence of a gene or cDNA that results in the amplification of a unique fragment (ie, a fragment that does not share more than 10 contiguous identical sequences with any other fragment on the microarray). It Computer programs well known in the art, such as
Oligo version 5.0 (National Biosciences) is useful for designing primers with the required specificity and optimal amplification properties. Typically, each probe on a microarray is between about 20 bases and about 50,000 bases in length, usually about
It will be between 300 and about 1,000 bases long. PCR methods are well known in the art,
For example, Innis et al., 1990, PCR Protocols: A Guide to Methods and Application
s, Academic Press, Inc., San Diego, California. As will be appreciated by those in the art, controlled robotic systems are useful for isolating and amplifying nucleic acids.

Another preferred means of purifying polynucleotide probes for microarrays for use in the methods and compositions of the invention is to synthesize synthetic polynucleotides or oligonucleotides using, for example, N-phosphonate or phosphoramidite chemistry. (Froehler et al., 1986, Nucleic Acid R
es. 14: 5399-5407; McBride et al., 1983, Tetrahedron Lett. 24: 246-248). Synthetic sequences are typically between about 4 and about 500 bases in length, more typically between about 4 and about 200 bases in length, and more preferably between about 15 and about 150 bases in length. In embodiments that use shorter oligonucleotide probes, synthetic nucleic acid sequences of less than about 40 bases in length are preferred, more preferably between about 15 and about 30 bases in length. In embodiments using longer oligonucleotide probes, the synthetic nucleic acid sequence is preferably between about 40 and 80 bases in length, more preferably between about 40 and 70 bases in length,
Even more preferably between about 50 and 60 bases in length. In certain embodiments, synthetic nucleic acids include, but are not limited to, unnatural bases such as inosine. As mentioned above, nucleic acid analogs may be used as binding sites for hybridization. Examples of suitable nucleic acid analogs are peptide nucleic acids (eg, Egholm et al., 1993.
, Nature 363: 566-568; U.S. Pat. No. 5,539,083).

In other embodiments, hybridization sites (ie, probes)
Are made from plasmids or phage clones of genes, cDNAs (eg expressed sequence tags), or inserts derived therefrom (eg Nguyen et al., 1995,
Genomics 29: 207-209).

Binding of Probes to Solid Surfaces Probes are preferably solid , eg made of glass, plastic (eg polypropylene, nylon), polyacrylamide, nitrocellulose, gels, or other porous or non-multi-antibiotics. It is attached to a phase support or surface. A preferred method of attaching nucleic acids to surfaces is Schena et al., 1995, Science 270: 467-47.
By printing on a glass plate, as outlined in 0. This method is particularly useful for preparing microarrays of cDNA (Derisi et al.
, 1996, Nature Genetics 14: 457-460; Shalon et al., 1996, Genome Res. 6: 639-64.
5; and Schena et al., 1995, Proc. Natl. Acad. Sci. USA 93: 10539-11286).

Yet another preferred method of making a microarray is by making a high density oligonucleotide array. Photolithographic techniques for in situ synthesis of arrays containing defined positions on the surface of thousands of oligonucleotides complementary to defined sequences (Fodor et al., 1991, Science 251: 767-773). ;
Pease et al., 1994, Proc. Natl. Acad, Sci, USA 91: 5022-5026; Lockhart et al., 1
996, Nature Biotechnology 14: 1675; U.S. Pat.No. 5,578,832; 5,556,752;
And No. 5,510,270) or other methods for rapid synthesis and deposition of defined oligonucleotides (Blanchard et al., Biosensors &
Bioelectronics 11: 687-690) is known in the art. When using these methods, oligonucleotides of known sequence (eg 25-mers) are synthesized directly on the surface of derivatized glass slides and the like. Usually, the arrays produced have several oligonucleotide molecules per RNA, with an extra part. Select an oligonucleotide probe to selectively splice a specific mRNA
Can also be detected.

For example, masking (Maskos and Southern, 1992, Nucl. Acids. Res. 20: 1679-
Other methods of making microarrays according to 1684) can also be used. In principle, and also as mentioned above, any type of array can be used, for example a dot blot on a nylon hybridization membrane (see Sambrook et al., Supra). However, as will be appreciated by those in the art, very small arrays will often be preferred due to the smaller amount of hybridization.

In a particularly preferred embodiment, the microarray used in the invention is provided by an inkjet printing device for oligonucleotide synthesis, eg
Blanchard, International Patent Publication No. WO98 / 41531, published September 24, 1998; Blanchar
d et al., 1996, Biosensors and Bioelectronics 11: 687-690; Blanchard, 1998, Sy.
nthetic DNA Arrays in Genetic Engineering, Vol. 20, JK Setlow, Plenu
m Press, New York, using methods and systems described on pages 111-123. In particular, oligonucleotide probes in such microarrays
It is preferably synthesized by sequentially depositing individual nucleotides for each probe sequence in an array of "microdrops" of a high tension solvent such as propylene carbonate. Microdroplets are small (eg 100 pL or less, more preferably 50 pL or less)
, Separated from each other (eg by hydrophobic domains) on the microarray to form circular surface tension wells that define the position of the elements (ie different probes) of the array.

Selection of Candidate Probes In a preferred embodiment, polynucleotide probes having a nucleotide sequence complementary to the nucleic acid sequence of a particular target polynucleotide are selected or provided by the method referred to herein as “tiling”. . Specifically, a polynucleotide probe having a nucleotide sequence of length 1 is selected by selecting a probe having a nucleotide sequence complementary to the sequence of 1 continuous bases of the target polynucleotide sequence. A polynucleotide probe is selected or provided, for example, by selecting or providing a polynucleotide probe having a nucleotide sequence complementary to 1 consecutive bases of the target polynucleotide sequence starting from the i-th base of the target polynucleotide sequence. can do. More specifically, the polynucleotide sequence is from bases i to i of the target polynucleotide sequence.
The first polynucleotide probe can be selected or provided by selecting or providing a polynucleotide probe complementary to the nucleotide sequence corresponding to +1. The second polynucleotide probe sequence is selected or provided by selecting or providing a polynucleotide probe whose nucleotide sequence is complementary to a nucleotide sequence corresponding to bases (i + n) to (i + n) +1, etc. of the target polynucleotide sequence. Can be provided.

As mentioned above, l specifies the length of the polynucleotide sequence of the probe. Therefore, 1 is a positive integer, preferably between about 4 and about 200, more preferably between about 15 and about 150. In embodiments using probes with shorter oligonucleotide sequences, 1 is preferably less than about 40, more preferably between about 15 and about 30. In embodiments using probes with longer oligonucleotide sequences, l is preferably about 4
It is between 0 and about 80, more preferably between about 40 and about 70, and even more preferably between about 50 and about 60.

The “tiling interval,” n, is a positive integer, preferably having a value between 1 and about 10. Particularly preferable values of the tiling interval include n = 1, 2, 3, 4 and 5. I, which is the starting position in the target polynucleotide sequence, is also a positive integer. In certain preferred embodiments, the starting position is 5 of the target polynucleotide sequence.
'-At or near the end. Therefore, i takes a preferred value of less than about 50, more preferably less than about 10. The first base in the target polynucleotide sequence is
This is a particularly preferred starting position in such an embodiment. Therefore, a particularly preferred value for the starting position is i = 1. In another preferred embodiment, only the 3'-end of the target polynucleotide sequence is tiled. For example, in some embodiments, only the last 2,000 bases at the 3'-end of the target polynucleotide sequence, more preferably only the last 1,000 bases, even more preferably the last 500 bases, even more preferably the last 350 bases. Only are tiled. In such an embodiment, the value of the starting position i is adjusted appropriately (e.g. i = L-2,000; i = L-1,000; i =
L-500; or i = L-350, where L is the length of the target polynucleotide sequence).

In certain embodiments, one or more probes to be evaluated are labeled, eg, maximally bound (ie, hybridized) to their target polynucleotide.
Further selection can be made by selecting only those probes that have or are predicted to have the energy ΔG. Methods of calculating or predicting the hybridization energy of a polynucleotide molecule are well known in the art, eg, a nearest neighbor model (eg Santa Lucia, 199).
8, Proc. Natl. Acad. Sci. USA 95: 1460-1465).
Those skilled in the art will readily adapt such models to various embodiments of the invention, including, for example, embodiments in which the polynucleotide molecule is immobilized on the surface of a solid support, such as in an array of polynucleotide probes. Can be done (eg 16 July 1999)
See Provisional Patent Application No. 60 / 144,382, filed on July 30; and US Patent Application No. 09 / 364,751, filed July 30, 1999). From such a model, for example Hynd
The binding energies can be readily evaluated using the mathematical algorithms and software described in man et al., 1996, Biotechniques 20: 1090-1096.

In such an embodiment, the binding energy can be calculated or predicted for each of a plurality of candidate probes for the target polynucleotide, such as the candidate probes selected by the tiling method described above. The probe predicted to have the highest binding energy is then selected for evaluation according to the method of the invention. Alternatively, probes predicted to have binding energies above a certain threshold (usually the threshold selected by the user) can be selected for evaluation according to the methods of the invention.

The above method is the preferred method of selecting polynucleotide probes regardless of the nature of the target polynucleotide sequence for which the probe is intended. In particular,
This method determines whether one or more target polynucleotides correspond to a unique gene (eg, one that does not have or is suspected of having any similar or homologous sequences in the sample), or one or more of the genes. It is preferred whether or not it is a member of the above families (eg one for which one or more analogs or homologues is known and / or is expected to be present in the sample). This method is also preferred regardless of the expected amount of target polynucleotide in the sample. Nevertheless, using the methods and compositions of the present invention,
It will be understood by those skilled in the art that the probe selected or provided by any method can be evaluated and is not limited to the embodiment in which the probe or probe sequence is selected according to the tiling method described above. Ah

In general, the probe selected for evaluation according to the methods of the invention will be a probe for only one particular target, but in at least some embodiments of the invention, a plurality of different targets ( Probes for, eg, two or more different polynucleotide sequences) may be simultaneously selected and evaluated using the methods and compositions of the invention. For example, in one embodiment of the invention, the Basic Local Alignm
The ent Search Tool (“BLAST”) or the PowerBLAST algorithm was used to express various polynucleotide sequences that did not contain sequences predicted or predicted to cross-hybridize each other's probes (eg, GenBank or dbEST databases, etc.). Can be identified (from within a sequence database). Such polynucleotide sequences are referred to herein as "orthogonal" sequences, and in embodiments where the polynucleotide sequences are sequences of a particular gene, are referred to as "orthogonal genes". According to the method of the present invention, different probes each hybridizing to different orthogonal sequences can be analyzed simultaneously so that the artifacts due to cross-hybridization by the gene-specific sample are minimized.

Algorithms for comparing polynucleotide sequences, such as the BLAST and PowerBLAST algorithms, are well known in the art (eg Altschul et al., 1990, J. Mol. Biol. 215: 403-410; Altschul, 1997, Nucleic Acids Res. 25: 3389-3402
And Zhang and Madden, 1997, Genome Res. 7: 649-656). Thus, those of skill in the relevant art will readily recognize how to use such algorithms to compare polynucleotide sequences, eg, using standard parameters well known in the art.

5.2.2. Hybridization Samples The methods and compositions of the present invention are referred to herein as first specific binding samples.
The amount or level of binding of each of the one or more probes of each sample to the amount or level of binding of each of the one or more probes of a second sample herein referred to as the non-specific binding sample. To characterize one or more probes. In a particularly preferred embodiment, the methods and compositions of the invention include binding (ie, hybridization) of a first sample, herein referred to as a specific hybridization sample, to each of one or more polynucleotide probes.
By comparing the amount or level with the amount or level of binding of a second sample, herein referred to as a non-specific hybridization sample, to each of the one or more polynucleotide probes, one or more Evaluate the properties of the polynucleotide probe.

The first sample (ie the specific hybridization sample) generally contains a particular target polynucleotide molecule, which is the intended target polynucleotide of the probe or probes to be evaluated. Alternatively, in an embodiment of the invention in which probes are evaluated for two or more different orthogonal target polynucleotide sequences, the specific hybridization sample comprises:
Preferred is a sample containing two or more different polynucleotide sequences. The target polynucleotide (or two or more orthogonal target polynucleotides) is preferably the amount of the target polynucleotide in the sample intended for the probe evaluated by the method of the invention (ie in the “true” sample), ie It is present in the specific hybridization sample in an amount that is comparable to or present in abundance. For example, in a preferred embodiment where the target polynucleotide corresponds to a gene or gene transcript expressed by a cell or organism, the target polynucleotide is preferably the amount or abundance of the target polynucleotide expressed by the cell or organism. In an amount that is comparable to
Present in the specific hybridization sample.

Most preferably, the one or more target polynucleotides are present in the specific hybridization sample in an amount or abundance equal to or abundant in the true sample. However, in many embodiments, the amount, or abundance, of the target polynucleotide in the true sample is unknown, or only in approximate amounts. Thus, in another embodiment, the target polynucleotide may be present in the specific hybridization sample in an amount or abundance approximately equal to that or abundance in the true sample. For example, the target polynucleotide may be present in the specific hybridization sample in an amount or abundance in the same order as that in the true sample or abundance.

Alternatively, the target polynucleotide is present in the specific hybridization sample in an amount or abundance between the minimum and maximum or abundance that can be expected for any one polynucleotide sequence in the true sample. You can For example, the amount or abundance of polynucleotide sequences typically in a true sample (ie, in a sample of polynucleotide molecules extracted from a cell or organism) is lower than that of poly A + mRNA extracted from a cell or organism. About 0.0001%, or at most about 3%
Can be. More preferably, the amount or abundance may be at most about 2%, more preferably about 1% of the poly A + mRNA extracted from the cell or organism. The amount or abundance of the polynucleotide sequence is more preferably about 0.0003% or more of the poly A + mRNA extracted from the cell or organism. Thus, for example, the target polynucleotide may be present in the specific hybridization sample in an amount equal to or approximately equal to the average or mean amount or abundance of the polynucleotides in the true sample. For example, in an embodiment in which the target polynucleotide corresponds to a gene or gene transcript of a cell or organism in which the target polynucleotide is present, the abundance or amount of the target polynucleotide in a specific hybridization sample is It may be equal to or approximately equal to the average abundance or amount of gene transcripts. Typical values for the average abundance or amount of a gene or gene transcript expressed in a cell or organism are known to those of skill in the art and generally depend on the cell or organism from which the gene or gene transcript is derived. . For example, typical preferred values include approximately 0.04% of all poly A + mRNA extracted from a cell or organism.

In other embodiments, the exact abundance or abundance of the target polynucleotide in the true sample may not be known, but its quantitative abundance will be known.
For example, in embodiments in which the target polynucleotide corresponds to a gene or gene transcript of a cell or organism where such gene or gene transcript is often at low or moderate levels, at moderate or high levels, or at high levels. Alternatively, it is characterized as being expressed in abundance. Thus, the target polynucleotide may be present in a specific hybridization sample in a typical or abundance of such quantitative abundances. One of ordinary skill in the art will readily recognize levels or abundance values for genes or gene transcripts in a sample that correspond to each of the above categories (ie, low, moderate, and high). In general, such values will depend on the particular cell type or organism from which the gene or gene transcript is derived. For example, a preferred value is about 1% or more of all polyA + mRNA extracted from cells for high levels or abundance, and extracted from cells for moderate levels or abundance. All poly A +
It is between about 0.01% and 1% of the mRNA and, for low levels or abundance, less than about 0.01% of all poly A + mRNA extracted from the cells.

The second sample (ie the non-specific hybridization sample) preferably contains a plurality of different polynucleotide molecules, each different polynucleotide molecule having a different polynucleotide sequence. In particular, in an embodiment of the invention where the sequence of the target polynucleotide is the sequence of a particular gene or gene transcript in a cell or organism, the nucleotide sequence of the polynucleotide molecule in the second, non-specific hybridization sample. Preferably contains sequences representing other genes or gene transcripts of the cell or organism.

Many different embodiments of specific and non-specific hybridization samples are possible. For example, in the first preferred embodiment, the specific hybridization sample is a substantially pure sample of molecules having a particular polynucleotide sequence. Preferably, these molecules are molecules of a particular gene or gene transcription (eg mRNA or cDNA) and thus have the sequence of that gene or gene product. The specific hybridization sample in this first embodiment should be at least 75% pure (i.e., 25% or less of the polynucleotide sequences in the sample are with sequences of the particular target polynucleotide or polynucleotides). different). Preferably, in this first embodiment, the specific hybridization sample is at least 90% pure, more preferably at least
95% pure, more preferably at least 99% pure.

It is understood that such specific hybridization samples can be readily prepared by those skilled in the art according to conventional methods now known in the art and without undue experimentation. For example, obtaining a polycleotide molecule corresponding to a particular gene or gene transcript, for example from genomic DNA, cDNA, mRNA (eg by RT-PCR) or cloned sequence by polymerase chain reaction (PCR) amplification of a gene segment. You can PCR primers are preferably selected based on the known sequence of the target polynucleotide (eg, of a particular gene or transcript of that gene) that results in the amplification of a unique fragment. As used herein,
Such “unique fragments” are fragments of a polynucleotide sequence that do not share more than 10 contiguous identical sequences with any other fragment in a PCR (or RT-PCR) sample. Oligo version 5.0 (National Biosciences) Computer programs well known in the art are useful and can be used in the design of primers with the required specificity and optimal amplification properties. PCR methods are well known in the art, for example, Innis et al., Eds., 1990, PCR Protocols: A Guide to Methods.
and Applications, Academic Press, Inc., San Diego, CA. One of ordinary skill in the art will appreciate that controlled robotic systems are useful for the isolation and amplification of nucleic acids containing target polynucleotides of interest to the user.

In another embodiment, the target polynucleotide in the specific hybridization sample is a plasmid or phage clone of the gene, a cDNA (eg, an expressed sequence tag), or an insert derived therefrom (eg, Nguyen et al., 1995). , Geno
mics 29: 207-209).

In this first embodiment, the second hybridization sample (ie the non-specific hybridization sample) contains a plurality of different polynucleotide molecules, each different polynucleotide molecule having a different nucleotide sequence. In particular, each of the nucleotide sequences of the polynucleotide molecules in the non-specific hybridization sample should be different from the nucleotide sequence of the target polynucleotide in the specific hybridization sample. For example, in embodiments where the sequence of the target polynucleotide is the sequence of a particular gene or gene transcript of a cell or organism, the nucleotide sequence of the polynucleotide molecule in the second, non-specific hybridization sample is preferably It includes sequences that represent other genes or gene transcripts of the cell or organism.

Such sequences are also preferably “for one or more probes intended”.
It is present in the non-specific hybridization sample at substantially the same abundance or amount as in the "true" sample (eg, in the cell or organism). In particular, the amount or abundance of each polynucleotide sequence in such a non-specific hybridization sample is preferably 100 times or less, more preferably 10 times or less, and more preferably the amount or abundance in the true sample. Different by 2 times or less, and even more preferably by 1.5 times or less (ie, 50% or less). However, in some instances, a few polynucleotides may be present in some cases, as typically observed, for example, in samples derived from mutated cells or organisms or samples exposed to one or more drugs. The relative amount or abundance of sequences (eg, preferably about 5% or less, more preferably about 1% or less, still more preferably about 0.1% or less) of different polynucleotide sequences in a non-specific hybridization sample is as described above. Can vary by more than the preferred amount of. However, the average amount of different polynucleotide sequences in a non-specific hybridization sample is preferably not substantially different from the average amount of different polynucleotide sequences in most typical "true" samples. More specifically, the average amount varies preferably by a factor of 2 or less, more preferably 50% or less, even more preferably 10% or less, most preferably 1% or less.

Most preferably, the cell or organism is currently known in the art, eg in
A cell or organism, such as E. coli or the yeast Saccharomyces cerevisiae, that can be manipulated according to routine techniques of in vitro homologous recombination and sexual genetics. In such an embodiment, the non-specific hybridization sample is, for example, a deletion of such cells or organisms in which the gene corresponding to the target polynucleotide sequence has been deleted or is silent (ie, not expressed in these cells). It can be prepared from the mutant.

Such non-specific hybridization samples also include mammalian cells or organisms (eg mouse, rat and human cells or organisms) for which convenient techniques such as in vitro homologous recombination and sexual genetics are not readily available. A) and cells. For example, a non-specific hybridization sample may be a strain in which a specific gene (in particular, a gene corresponding to a target polynucleotide) is deleted, or a mammalian cell (for example, mouse, rat or human) which becomes available. It can also be prepared from obligate diploids such as cell cultures including cell cultures.

Methods of preparing polynucleotide samples from such deletion mutants are well known in the art. For example, methods of preparing whole poly (A) ⁺ from cells or organisms are well known in the art and are generally described by Sambrook et al., Eds., 1989, Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Sp
described in ring Harbor, New York. In one embodiment, RNA is extracted from various types of cells of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al., 1979, Biochemistry 18: 5294-5299). S
In another preferred embodiment for S. cerevisiae, Ausubel et al. (Edited by Ausubel et al., 198
9, Current Protocols in Molecular Biology, Vol.III, Green Publishing Ass
ociates, Inc., John Wiley & Sons, Inc., New York, pp.13.12.1-13.12.5), RNA is extracted from cells using phenol and chloroform.
Poly (A) ⁺ RNA is selected by selection with oligo-dT cellulose.

In one embodiment, RNA can be fragmented by methods known in the art, such as incubation with ZnCl2. In one embodiment, isolated mRNA can be converted to antisense RNA synthesized by in vitro transcription of double-stranded cDNA in the presence of labeled dNTPs (Lockhart et al., 1996, Nature Biotechnol.
ogy 14: 1675).

In another embodiment, the polynucleotide molecule of the non-specific hybridization sample is fragmented genomic DNA, first strand cDNA reverse transcribed from mRNA, or amplified mRNA or a PCR product of cDNA, or the like. Contains DNA molecules. Methods of preparing such samples are also well known in the art and one of ordinary skill in the art will readily recognize how to prepare such non-specific hybridization samples without undue experimentation.

Alternatively, in embodiments where deletion mutants of cells or organisms are not readily available, non-specific hybridization samples can also be prepared from the polynucleotide mixture with the target polynucleotides removed. . For example, a non-specific hybridization sample may be one or more libraries of selected clones that do not contain one or more clones corresponding to the target polynucleotide, or the clones corresponding to the target polynucleotide have been removed. It can be prepared from The non-specific hybridization sample may also be subtractively hybridized with the gene of interest (ie the gene corresponding to the target polynucleotide) to remove the gene from the sample, or at least reduce its amount to partially remove it. It can also be prepared from a DNA or mRNA sample prepared from cells such as.

In a second preferred embodiment of the invention, the specific hybridization sample is the same as the specific hybridization sample of the first preferred embodiment above. However, the non-specific hybridization sample of this second embodiment is preferably derived from a source containing a normal amount of the target polynucleotide in addition to a plurality of other polynucleotide sequences. For example, in embodiments where the sequence of the target polynucleotide is the sequence of a particular gene or gene transcript of a cell or organism, the nucleotide sequence of the polynucleotide molecule in the second, non-specific hybridization sample is preferably It includes sequences that show both the gene or gene transcript corresponding to a particular target polynucleotide and other genes or gene transcripts of the cell or organism.

Such non-specific hybridization samples may be normal or “wild type” cells or organisms that express normal amounts of the gene or gene transcript corresponding to the target polynucleotide sequence, as well as other genes or gene transcripts. Prepared using the method for extracting a polynucleotide from a cell or organism as described above for the preparation of a non-specific hybridization sample in the first particularly preferred embodiment of the invention, which is well known in the art. can do. Therefore,
This second embodiment is particularly preferred in aspects of the invention where the target polynucleotide corresponds to a gene or gene transcript of a cell or organism for which deletion mutants are not readily available, for example. The level of non-specific hybridization is determined according to the following method of the invention, for example by subtracting the hybridization signal obtained from the specific hybridization sample from the hybridization signal obtained from the non-specific hybridization sample,
It can be evaluated from such samples.

In a third preferred embodiment of the invention, the specific hybridization sample may contain not only the target polynucleotide sequence, but also other non-target polynucleotide sequences. For example, in an embodiment of the invention where the sequence of the target polynucleotide is the sequence of a particular gene or gene transcript of a cell or organism,
The first, the nucleotide sequence of the polynucleotide molecule in the specific hybridization sample may include the polynucleotide sequence corresponding to both the target polynucleotide and other genes or gene transcripts of the cell or organism. Thus, in such a third embodiment, the specific hybridization sample is preferably the same as the non-specific hybridization sample described above for the second preferred embodiment of the invention. In particular, the specific hybridization sample in this third embodiment of the invention is most preferably the gene or gene transcript of the target polynucleotide, as well as other genes or gene transcripts at normal levels in the cell or organism. A polynucleotide sample obtained from expressing normal or wild type cells or organisms.

The second or non-specific hybridization sample in this third preferred embodiment of the invention is preferably the same as the non-hybridization sample described above for the first preferred embodiment of the invention. In particular, this third
The non-specific hybridization in a preferred embodiment of preferably comprises a plurality of different nucleotide molecules, each different polynucleotide molecule having a different polynucleotide sequence, each polynucleotide sequence in the non-specific hybridization sample being targeted. It differs from the polynucleotide sequence of the polynucleotide. For example, in a particularly preferred aspect of this third embodiment, the non-specific hybridization sample is obtained from a deletion mutant of a cell or organism in which the gene corresponding to the target polynucleotide sequence is deleted or silent. It is a polynucleotide sample. Such an embodiment is particularly preferred in applications where it is important to assess the specificity and / or sensitivity of the probe, eg in the natural amount of the target polynucleotide.

In a fourth preferred embodiment of the invention, both the specific hybridization sample and the non-specific hybridization sample are (a) a polynucleotide molecule having the polynucleotide sequence of the target polynucleotide, and (b) A plurality of different polynucleotide molecules, each different polynucleotide molecule having a different polynucleotide sequence different from the sequence of the target polynucleotide. In this fourth embodiment, the amount or level of target polynucleotide molecules in the first or specific hybridization sample is substantially the same as the amount or level of target polynucleotide molecules in the second or non-specific hybridization sample. Differently. Specifically, the amount or level of target polynucleotide molecules differ by preferably at least 2-fold, more preferably at least 4-fold, even more preferably at least 8-fold, even more preferably at least 20-fold, even more preferably at least 100-fold.

In this fourth embodiment, preferably the different polynucleotide molecules (ie polynucleotide molecules having a sequence different from the target polynucleotide sequence) are substantially identical in both the specific and non-specific hybridization samples. Is. In particular, each of the other "non-target" polynucleotide sequences is preferably present in substantially equal amounts or abundances in both samples, and these amounts or abundances are in the "true" sample ( The amount or abundance of polynucleotide sequences (eg, in a sample of polynucleotides of cell or organism origin) is preferably substantially the same. Specifically, the amount, or abundance, of each non-target polynucleotide molecule is preferably 100 times or less, more preferably 10 times or less, still more preferably 2 times or less, still more preferably 1.5 times between two hybridization samples. Less than double (ie less than 50%). However, between two hybridization samples and / or between hybridization samples, as typically found in samples from mutant cells or organisms, or cells or organisms treated with one or more drugs, for example. And the true sample has a greater change in the relative amount of 2-3 polynucleotide sequences (eg, preferably about 5% or less, more preferably about 1% or less of the different polynucleotide sequences in the non-specific hybridization sample). , And more preferably about 0.1% or less). However, the abundance or average change in the abundance or amount of all non-target polynucleotide sequences is preferably less than 2-fold, more preferably less than 50%, even more preferably less than 10%, even more preferably less than 1%.

[0111]   Specific hybridization sample and non-specific hybridization sample
Both polynucleotide molecules of the sample are preferably detectably labeled. Preferred
Preferably, the detectable label is a fluorescent label, for example by incorporation of nucleotide analogues.
Knowledge. Other preferred labels for use in the present invention include, but are not limited to
But not biotin, iminobiotin, antigen, cofactor, dinitrophenol
, Lipoic acid, olefinic compounds, detectable polypeptides, electron-rich molecules, substrates
And an isotope capable of producing a detectable signal by its action on
Is mentioned. Preferred radioisotopes include a few³²P, ³⁵ S,¹⁴C,¹⁵N, and¹²⁵I is mentioned. Fluorescent molecule suitable for the present invention
Include, but are not limited to, fluorescein and its derivatives, rhodami
And its derivatives, Texas Red, 5'-carboxy-fluorescein ("FMA
)), 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxy-fluorescein ("J
OE "), N, N, N ', N'-tetramethyl-6-carboxy-rhodamine (" TAMRA ")
), 6'-carboxy-X-rhodamine ("ROX"), HEX, TET, IRD40
And IRD41. Further, the fluorescent molecule suitable for the present invention is further limited to
Cyamine dyes, including but not limited to Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and FluorX, limited
BODIPY-FL, BODIPY-TR, BODIPY-TMR, BODIPY-630 / 650, and BODIPY
-BODIPY dyes including 650/670, including but not limited to ALEXA-488, ALEXA-532, ALEXA-54
ALEXA dyes, including 6, ALEXA-568, and ALEXA-594, and other fluorescence known to those of skill in the art.
Further examples are dyes. Suitable electron-rich indicator molecules in the present invention include
, But not limited to, aferritin, hemocyanin, and colloids (coll
iodal) Gold is mentioned. Alternatively, in some embodiments, the target polynucleotide
Otide may be used to specifically complex the first group to the polynucleotide.
Can be labeled with and. Covalently attached to the indicator molecule, the first group
Indirect detection of target polynucleotide using a second group with affinity for
can do. In such embodiments, it is suitable for use as the first group.
Suitable compounds include, but are not limited to, biotin and iminobiotin.
Can be mentioned.

In a particularly preferred embodiment, the two samples (ie the specific hybridization sample and the non-specific hybridization sample) are distinguishably labeled. Specifically, each sample is labeled with a different detectable label (eg, with a different individual fluorophore), and by detecting the label of each sample, two samples can be detected simultaneously and can be distinguished from each other. To do so. For example,
In one embodiment, specific hybridization samples can be labeled with fluorescein labeled dNTPs that fluoresce green while non-specific hybridization samples can be labeled with rhodamine labeled dNTPs that fluoresce red. Each sample can then be easily detected and distinguished from other samples by detecting the characteristic green or red fluorescence of the label of each sample.

In fact, by using such a differential label, two hybridization samples can be distinguished from each other when mixed,
It can be detected separately. Thus, specific and non-specific hybridization samples need not be kept separate from each other, but can be mixed and hybridized with one or more polynucleotide probes, eg on the same microarray and in the same experiment at the same time. You can Therefore, it is not essential for the present invention that the specific hybridization sample and the non-specific hybridization sample are physically separated samples. The two samples are then, for example, by means of the identification labeling scheme above.
It only needs to be identifiable. Alternatively, one of skill in the art will appreciate that in such embodiments, the specific hybridization sample and the non-specific hybridization sample may comprise two different distinct (eg, distinguishably labeled) components: (specific hybridization as described above). It will be readily appreciated that the same sample with a specific hybridization component (corresponding to the sample) and a non-specific hybridization component (corresponding to the non-specific hybridization sample described above) can be considered.

5.2.3 Determining Hybridization Levels The properties of one or more probes can be characterized according to the methods and compositions of the invention by determining the amount or level of binding of the first specific binding sample to the probe and the second non-binding. Specific binding samples are assessed by comparing the amount or level of binding to the probe. In a preferred embodiment in which the probe and target comprise a polynucleotide molecule, the amount or level of hybridization of the first specific hybridization sample to the polynucleotide probe is changed to the polynucleotide probe of the second non-specific hybridization sample. Compare with the amount or level of hybridization. Thus, the method of the present invention preferably comprises the step of obtaining or providing a level of hybridization to the pronucleotide probe at the level of specific hybridization sample and non-specific hybridization sample, eg, the polynucleotide in the sample. The two samples are contacted with the polynucleotide probe under conditions such that the molecule binds to or hybridizes with the molecule of the probe, and the polys from each of the two samples that bind or hybridize with the molecule of the probe Measuring the amount of nucleotides.

Hybridization Conditions The conditions under which a polynucleotide is contacted with a probe are known in the art as "hybridization conditions." Preferably, the hybridization conditions are such that there is high specific binding of the polynucleotide molecule to the probe (eg binding of the polynucleotide molecule from a specific hybridization sample),
Non-specific binding of the polynucleotide molecule to the probe (eg, binding of the polynucleotide molecule from the non-specific hybridization sample) is optimized. However, in some embodiments the optimal hybridization conditions are not known, but only roughly. For example, in certain embodiments, the methods and compositions of the invention can be used to evaluate particular hybridization conditions, eg, to determine if hybridization conditions are optimal. For example, one or more hybridization parameters (eg, temperature and / or salt concentration) can be intentionally varied, and the method of the invention can be used, for example, to determine the specificity of the probe for each hybridization condition and / or Or it can be used to determine sensitivity. Suitable or preferred hybridization conditions are then determined as the hybridization conditions that maximize the sensitivity and / or specificity of the probe.

In a particular embodiment in which the probe comprises a double-stranded DNA sequence, the probe or an array comprising those probes is preferably subjected to denaturing conditions to obtain a DNA single strand prior to contacting with the target polynucleotide molecule. Be done. Arrays containing single-stranded probe DNA (eg, synthetic oligodeoxyribonucleic acid) are also denatured before contacting the target polynucleotide molecule, eg, to remove hairpins and dimers formed due to self-complementary sequences. It will be necessary.

Optimal hybridization conditions include the length (eg, oligomer for a polynucleotide of 200 bases or more) and type (eg, RNA) of the probe and the target nucleic acid.
Or DNA). Specific (ie, stringent)
General parameters for hybridization conditions are described, for example, in Sambrook et al., E.
ds 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Har
bor Laboratory Press, Cold Spring Harbor, New York, p. 9.47-9.51 and
11.55-11.61; and Ausbel et al., 1987, Current Protocols in Molecular Bio.
logy, Greene Publishing and Wiley-Interscience, New York. In embodiments where a CDNA microarray is used, typical hybridization conditions are 65 ° C. for 4 hours in 5 × SSC and 0.2% SDS, followed by low stringency wash buffer (1 × SSC and 0.2% SDS). Wash at 25 ° C., followed by 10 min at 25 ° C. in higher stringent wash buffer (0.1 × SSC and 0.2% SDS) (Shene et al., 1996, Proc. Natl. Acad. Sci. USA). 93: 10614).
Useful hybridization conditions can also be found in, for example, Tijessen, 1993, Hybridizat.
ion With Nucleic Acid Probes, Elsevier Science Publishers; BV and Kri
Also provided by cka, 1992, Nonisotopic DNA Probe Technique, Academic Press, San Diego.

In preferred embodiments in which an oligonucleotide probe is used, preferred hybridization conditions are: 1 M NaC, 50 mM MES buffer pH 6.5, 0.5% sarcosine sodium and 32% formamide, at the average melting temperature of the probe or Hybridization at near temperatures (eg, within 5 ° C., more preferably within 2 ° C.) can be included.

The target polynucleotide melting temperature (Tm) from a particular probe is known in the art as the temperature at which half (ie, 50%) of the target polynucleotide molecules in a sample bind to the probe molecule. ing. The term is used herein to describe and enable suitable hybridization conditions, such that the melting temperature of the probe has a nucleic acid sequence that is complementary to the nucleic acid sequence of the probe, the target polynucleotide molecule in the sample. ½ means the temperature at which the probe binds. Methods for determining the melting temperature of a particular polynucleotide duplex are well known in the art, for example there is a method of predicting the melting temperature using well-known physical models fitted to experimental data. (SantaLucia, 1998, Proc. Natl. Acad. Sci.
USA 95: 11460-11465 and its references). Mathematical algorithms and software for melting temperature prediction used in such models are readily available, for example, as described in Hyndman et al., 1996, Biotechniques 20: 1090-1096. The specific parameters used in such models are usually derived for the polynucleotide duplexes in solution, but the appropriate parameters were readily obtained, eg immobilized on a solid phase in a microarray. The melting temperature and other hybridization characteristics of the target polynucleotide that hybridizes to the polynucleotide probe can be predicted. In fact, one of ordinary skill in the art would, as described in Stoughton, et al. It is easy to know whether to obtain the appropriate parameters for the specific microarray probe. Melting temperatures for RNA / DNA duplexes of about 25 base pairs in 1M salt solution are typically about 60 ° C and about 7 ° C.
It is between 0 ° C.

Signal Detection Hybridization conditions used in the methods of the invention, including the specific hybridizations described above, also include wash conditions. Washing conditions are preferably such that polynucleotide molecules that do not bind to the probe are removed (eg, from the probe microarray), but the bound probe and polynucleotide molecules remain. The amount of polynucleotide hybridized to each probe can then be measured and determined, eg, by measuring or determining the amount of detectable label.

As mentioned above, preferably polynucleotides from two different samples (eg, from a specific hybridization sample and a non-specific hybridization sample) hybridize to the probe simultaneously. For example, in a preferred embodiment of the invention where a number of different probes on the microarray are evaluated, the two samples hybridize at the same time to the binding sites on the microarray. In such an embodiment, the polynucleotide samples from each of the two samples are separately labeled so that they can be distinguished. For example, the cDNA in a specific hybridization sample can be labeled with fluorescein-labeled dNTPs, and the cDNA from a non-specific hybridization sample can be labeled with a rhodamine-labeled dN.
It can be labeled with TP. When two cDNAs are mixed and hybridized to a microarray, the relative intensities of the signals from each set of cDNAs are measured at each site on the array, thereby detecting the relative difference in the amount of a particular mRNA. It

In the above example, the cDNA from the specific hybridization sample fluoresces green when stimulated with a fluorescent dye (eg, fluorescein label),
The cDNA from the non-specific hybridization sample fluoresces red. Consequently, when a particular probe (eg, on a microarray) specifically hybridizes to a particular target polynucleotide (eg, a target polynucleotide of a specific hybridization sample), that probe on the microarray The binding site of emits light at a wavelength characteristic of fluorescein labeling (eg,
green). In contrast, when the probes on the microarray cross-hybridize to other polynucleotides (eg, from a non-specific hybridization sample), the binding sites on the microarray of the probes are of wavelengths characteristic of both labels. Emits light. A probe that specifically hybridizes to a target polynucleotide of a specific hybridization sample emits green fluorescence at a higher ratio to red fluorescence, whereas a probe that hybridizes to the target polynucleotide with weaker specificity. Emits green fluorescence at a lower ratio to red fluorescence.

The use of such two-color fluorescent labels and detection schemes has been described, for example to identify differences in gene expression (see Shene et al., 1995, Science 270: 467-470). The advantage of using cDNA labeled with two different fluorescent dyes is that it allows a direct and internally controlled comparison of hybridization levels corresponding to specific and non-specific hybridization. Changes due to small differences in experimental conditions (eg, hybridization conditions) will not affect subsequent analysis. However, the present invention also uses two physically separated samples, eg, the absolute amount of cDNA or mRNA (or other polynucleotide) from a specific hybridization sample that hybridizes to the probe and the same probe. It will be appreciated that it can also be done by comparing the absolute amounts of cDNA or mRNA from hybridizing non-specific hybridization samples.

When fluorescently labeled targets are used, the fluorescence emission at each site of the microarray can be detected, preferably by scanning with a confocal laser microscope. In one embodiment, a separate scan with appropriate excitation lines is performed for each of the two fluorochromes used. Alternatively, a laser capable of simultaneously irradiating two fluorescent dyes at specific wavelengths can be used, and the emission from the two fluorescent dyes can be analyzed simultaneously (Shena et al., 1996, Genome Res. 6: 639- See 645). In a preferred embodiment, the array is a computer controlled XY
Scanned with a laser fluorescence scanner with stage and microscope objective. Sequential excitation of the two fluorochromes is achieved using a multi-line mixed gas laser and the emissions are wavelength separated and detected in two photoamplifier tubes. Such fluorescence laser scanning devices are described, for example, in Schena et al., 1996, Genome Res. 6: 639.
-645. Alternatively, Ferguson et al., 1996, Nature Biotech. 14: 1681.
The bundle of fiber optics described in -1684 can be used simultaneously for hybridization levels at multiple binding sites.

The signals are recorded and, in a preferred embodiment, computer analyzed, for example using a 12-bit analog to digital board. In one embodiment,
The scanned image is a graphics program (eg Hijaak Graphics
Suite) and then analyzed using an image gridding program that produces a spreadsheet of average hybridizations at each wavelength at each site. If necessary, an experimentally determined correction for "cross talk" (ie, overlap) between the two fluorochrome channels is made. At any particular hybridization site on the microarray, the ratio of emission of the two fluorochromes can be calculated. The ratio does not depend on the absolute amount or level of hybridization of either sample, but is specific and useful in determining the relative amount of cross-hybridization from the two samples, as described above.

5.2.4. Data Analysis The methods and compositions of the present invention are useful for assessing one or more probes, and more specifically, using them to detect one or more probes. The sensitivity and / or specificity of binding to or hybridizing to a particular target can be assessed. The term sensitivity of a probe, as used herein, is understood to refer to the absolute amount or level of a particular target that binds to the probe under particular binding conditions (ie the number of molecules of the particular target). . The amount or level of a particular target that binds to a probe under certain binding conditions is also referred to herein as the specific binding amount or level of specific binding of the probe under certain binding conditions. In a preferred embodiment of the invention where the probe and target are polynucleotide molecules, the sensitivity of the probe (ie polynucleotide probe) is determined by the absolute amount of a particular target polynucleotide that hybridizes to the polynucleotide probe under particular hybridization conditions. (Ie, the number of polynucleotide molecules having a nucleotide sequence).
The amount of a particular target polynucleotide that hybridizes to a probe under particular hybridization conditions is also referred to herein as the amount of specific hybridization of the probe under the particular hybridization conditions.

The term specificity of a probe, as used herein, refers to the amount of non-specific binding or level of non-specific binding of the probe to the probe under the same binding conditions under the specific binding conditions. Amount or level of specific target bound (ie number of molecules of specific target)
Is understood to mean. The term non-specific binding, as used herein, refers to the amount of molecules other than said specific target molecule that bind to a probe under specific binding conditions (ie the number of molecules that are not said specific target molecule). To be understood. In a preferred embodiment of the invention in which the probe and target are polynucleotide molecules, the sensitivity of the probe is the same as, or compared to, the amount that cross hybridizes to the probe under specific hybridization conditions. Is understood to refer to the amount of a specific target polynucleotide that hybridizes with the probe under the hybridization conditions (i.e., the number of polynucleotide molecules having a specific nucleotide sequence). The term cross-hybridization or non-specific hybridization as used in a preferred embodiment of the present invention refers to the amount of a polynucleotide other than a specific target polynucleotide that hybridizes to a probe under specific hybridization conditions (i.e. It is understood to refer to the number of polynucleotide molecules that have a nucleotide sequence that differs from the nucleotide sequence of the target polynucleotide.

In the methods and compositions of the present invention, the specific hybridization T of the target polynucleotide to the probe p is directly related to the intensity I of the hybridization signal from the specific hybridization sample. This relationship is for example expressed by the following equation: T _p = s · I _p ^S (Equation 1) where I _p ^S is the hybridization at probe p to the specific hybridization sample (ie the sample containing the target polynucleotide sequence). Is the intensity of the signal, s is a correction factor such as for the efficiency of the detector or label. ] Can easily represent.

Similarly, the amount of cross-hybridization X with the probe p is directly calculated by the following formula: X _p = s · I _p ^NS (Formula 2) [wherein I _p ^NS is non-specific hybridization] It is the intensity of the hybridization signal at probe p relative to the sample (ie the sample with the target polynucleotide removed). ] Relates to the intensity of the hybridization signal from the non-specific hybridization sample.

[0130]   Therefore, one of ordinary skill in the art would readily appreciate the hybridization intensity I from the two samples._p ^S And I_p ^NSEasily determine the sensitivity and specificity of a particular probe by comparing
Understand that it is determinable. In a particularly preferred embodiment, such a comparison
Includes (specific hybridization from hybridization sample
Intensity) vs. (hybridization from non-specific hybridization samples)
Intensity) ratio (ie ratio I_p ^S/ I_p ^NS) Or obtaining the ratio.
Specifically, as mentioned above, the specificity of the probe depends on the probe under certain conditions.
The same conditions for the amount of non-specific binding or the amount of non-specific hybridization
Defined as the amount of a particular target that binds or hybridizes to the probe under
You can Therefore, in one embodiment, the specificity S of the probe p is
formula:       S_p= T_p/ X_p            (Formula 3) Obtained by

However, from equations 1 and 2 above, the specificity is also given by the equation: S _p = I _p ^S / I _p ^NS (equation 4), which will be readily apparent to the ^{skilled person} or Obtainable.

Thus, the ratio of hybridization intensities of specific and non-specific hybridization samples provides a measure or value for the specificity of the probe.

Similarly, in the methods and compositions of the invention, the sensitivity of probe p is determined or obtained from the hybridization intensity of the specific hybridization sample for the probe, ie, I _p ^S. Generally, the sensitivity of the probe will correlate with the specificity of the probe. Therefore, in a preferred embodiment, such probes that are more specific to the target polynucleotide will be more sensitive to the target polynucleotide.

The analysis method of the present invention is preferably carried out by a computer system as described below. FIG. 7 shows an example of a computer system suitable for carrying out the analysis method of the present invention. A computer system, such as the exemplary computer system 701, consists of one or more internal components and is connected to one or more external components. Internal components of such a computer system include a processor element 702 interconnected with a memory 703. A specific example of a computer system may be an Intel Pentium-based processor with a clock speed of 200MHz or higher and a main memory of 32MB or higher.

External components include a mass storage 704. The mass storage may be, for example, one or more hard disks, which are typically packaged with the processor and memory. The hard disk typically has a storage capacity of 1 GB or more. Other external components include one or more user interface devices 705, which may include, for example, a monitor, a keyboard with a pointing device 706 (eg, a "mouse"), or other character input device. Generally, the computer system is further connected to a network link 707, which may be, for example, one or more other terminal computer systems, one or more remote computer systems, or one or more wide area communication networks (eg, the Internet).
Can be part of an Ethernet connection to. Such a network connection enables the computer system to share data and processing work with other computer systems.

During operation of the above system, the memory is loaded with several software elements that are standard in the art and are special to the present invention. Together, these software elements cause the computer system to function according to the methods of the present invention (ie, the computer system causes a processor to perform the methods of the present invention). Software elements are typically computer-readable media (
For example, coded and stored on the mass storage component 704). However, one or more of the software elements may be encoded and stored on other forms of computer readable media. Such media include, but are not limited to, floppy disks, CD-ROMs or DAT tapes. Software element 710 represents the operating system responsible for managing the computer system and its network interconnections. This operating system is, for example, the Microsoft Windows® family (eg Windows 95, Window
s 98, Windows 2000 or Windows NT). Alternatively, the operating system may be a Macintosh operating system or a Unix operating system (eg LINUX). Software elements 711 represent common languages and functions conveniently present in the system and assist programs implementing the particular methods of the present invention. Languages that can be used to program the parsing methods of the present invention include, for example, C, C ++, and less preferred than this, FORTR.
Also includes AN and JAVA. Most preferably, the method of the present invention is programmed in a mathematical software package. This package enables a high level specification of symbolic mathematical expression capture and processing, including the particular algorithm to be used, thereby eliminating the need for the user to procedurally program individual mathematical expressions and algorithms. Such packages include, for example, Matlab from Mathworks (Natick, Mass.), Wolfram Research (Shapain, IL).
) Mathematic, or Math Soft, Inc. (Seattle, WA) S-Plus.
Accordingly, software element 712 represents the parsing method of the present invention as programmed into a procedural language or symbol package. The software element can also include an element 713 containing data used in the analysis method of the present invention, for example in a database. For example, a database element can include data that indicates the amount that a molecule in one or more samples binds (eg, hybridizes) to one or more probes. Such computer systems can be used to implement and carry out the methods of the invention. In particular, the user can execute the analysis software element 712 of the system, which allows the processor to carry out the method of the invention and evaluate the binding of one or more probes to one or more different target molecules.

The compositions of the present invention also include computer program products. This product can be used to load one or more of the above software elements into the memory of a computer system, and to cause a processor of the computer system to carry out the method of the present invention. Such computer program products typically include one or more computer-readable storage media (eg, floppy disk, CD-ROM, DAT tape, etc.) in which one or more computer program mechanisms are embedded or coded. Include. In particular, the computer program mechanism may, for example, enable the program mechanism to be loaded into a memory of a computer system (eg, the memory of the exemplary computer system 701) and cause the processor of the computer system to perform the analysis methods of the present invention. Included are one or more of the above software components.

Since alternative systems and methods for carrying out the analytical methods of the present invention will also be recognized by those of ordinary skill in the art, such systems and methods should be understood within the scope of the appended claims. . For example, one of ordinary skill in the art will recognize alternative program structures that can be used, for example, in a computer system or computer program product to implement the methods of the present invention. Therefore, it is understood that systems and products including such alternative program structures are also part of the present invention.

5.3. Application to Probe and Microarray Design The methods and compositions of the present invention are particularly useful in the design of microarrays, which have many applications, such as in the fields of biology and drug discovery. For example, using the methods and compositions of the invention, a probe having the ability to screen and specifically detect a large number of different target polynucleotides, such as a large number of different genes or gene transcripts in a cell or organism. Microarrays can be made.

For example, what is referred to herein as such a “screening chip” is capable of specifically hybridizing to at least 2,000, or at least 4,000 different target polynucleotides and thus detecting them. Can include a probe. More preferably, such screening chips have probes capable of specifically hybridizing to and detecting at least 10,000, at least 15,000, or at least 20,000 different target polynucleotides. In a particularly preferred embodiment, a screening chip having more than 50,000 types, more than 80,000 types, or more than 100,000 types specifically different hybridizing with different target polynucleotides, as a result having a probe capable of detecting them. Can be made.

In an embodiment where the screening chip is used for the detection of a polynucleotide corresponding to a gene or gene transcript of a cell or organism, therefore,
Such screening chips will typically have a probe that specifically and discriminately hybridizes to at least 50% of the genes in the genome of the cell or organism. More preferably, the screening chip is at least 75%, at least 80%, at least 85%, at least 9% of the genes in the genome of the cell or organism.
It may have a probe that specifically and distinguishably hybridizes with 0%, at least 95%, or at least 99%. Indeed, in a particularly preferred embodiment, the screening chip comprises a probe that hybridizes specifically and distinguishably with all (ie 100%) of the genes in the genome of the cell or organism.

The methods and compositions of the invention can also be used to generate microarrays of probes that have the ability to specifically detect a small number of different target polynucleotides with greater sensitivity and specificity. For example, using the method of the present invention, “herein
`` Signature gene, '' for example, can be used to reliably and accurately identify changes in a particular gene that change in response to a perturbation of some change to a cell or organism that expresses or potentially expresses those genes. A detectable microarray can be designed.

Specifically, the methods described above can be used to simultaneously determine the sensitivity and specificity of multiple different probes. Then rank the probes (eg, US Provisional Application No. 60 / 144,382, filed July 16, 1999 and July 1999).
(According to the method described in U.S. patent application Ser.No. 09 / 364,751 filed on 30th), and then selecting from that plurality the particular probe that is both the most sensitive and specific for a particular target. Select. Such probes can then be used in screening arrays and signature arrays, or microarrays such as the "chips" described above.

In particular, by using probes optimized for both sensitivity and specificity, the number of different probes required for reliable and distinguishable detection of a particular polynucleotide is significantly reduced. (Eg, as few as one probe for each target polynucleotide). In this way, microarrays can be produced that have probes that hybridize specifically and recognizable to many different polynucleotides. Alternatively, a microarray capable of detecting the amount or level of a particular target polynucleotide in one sample with higher reliability and accuracy using a probe optimized for both sensitivity and specificity for a particular target, Alternatively, microarrays can be made that can detect changes in the amount or level of a particular target polynucleotide in two or more different samples with greater confidence and accuracy. Therefore, such microarrays will typically have binding sites (ie, probes) that specifically and discriminately bind to a small number of different polynucleotides.

The method of the invention involves selecting probes for a plurality of different target polynucleotides by one of ordinary skill in the art, for example, by repeating the above method for each particular target polynucleotide in the species. Can be easily applied to. Alternatively, in certain embodiments, the methods of the invention can be used to evaluate and select probes for two or more different polynucleotides. In this case, the two or more different polynucleotides are sufficiently orthogonal to each other that they do not cross hybridize to a common polynucleotide sequence. "Orthogonal" polynucleotides mean two or more polynucleotides that do not contain a common nucleic acid sequence. Therefore, orthogonal sequences are not expected to cross hybridize. In particular, when the first polynucleotide sequence and the second polynucleotide sequence are orthogonal sequences, the complementary sequence that hybridizes to the first polynucleotide sequence does not hybridize to the second polynucleotide sequence. Unthinkable. Thus, more specifically, any two or more different target polynucleotide molecules used in such alternative embodiments may be any of the other different target polynucleotide molecules used in this embodiment. A probe that hybridizes to or cross-hybridizes with
Will not hybridize or cross hybridize.

Sequences that are sufficiently orthogonal for use in such embodiments of the invention are sequences that have less than 50% sequence identity with each other, and more preferably less than 20%, less than 10% of each other. , Less than 5%, less than 2%, or less than 1% sequence identity. Such fully orthogonal sequences are, for example, sequence comparison algorithms (Basic Local Alignm for identifying such sequences in nucleotide sequence databases (e.g. in GenBank or dbEST databases)).
ent Search Tool (BLAST) or Power BLAST algorithm). Sequence comparison algorithms such as BLAST and Power BLAST are well known in the art (eg Altschul et al., 1990, J. Mol. Biol. 215: 403-410; Altsc.
hul, 1997, Nucleic Acids Res. 25: 3389-3402; and Zhang and Madden, 199.
7, Genome Res. 7: 649-656). Accordingly, one of ordinary skill in the art will readily appreciate that such algorithms can be used to compare polynucleotide sequences using, for example, standard parameters well known in the art.

The methods of the invention can also be used to test theoretical models that predict properties such as sensitivity and specificity of polynucleotide probes. For example, US Provisional Application No. 60 / 144,382, filed July 16, 1999, and US Patent Application No. 09 / 364,751, filed July 30, 1999, describe the susceptibility of certain oligonucleotides. And a method that can be used to calculate or predict specificity, such as an adjacency model (e.g., S
antaLucia, 1998, Proc. Natl. Acad. Sci. USA 95: 1460-1465), and theoretical models of hybridization thermodynamics of polynucleotides are disclosed. Using the methods and compositions of the invention disclosed herein, the hybridization properties (such as sensitivity and specificity) of one or more probes can be empirically determined, and can also be determined, for example, by a theoretical model as described above. Can be compared with the value predicted by. Such comparisons can then be used, for example, to test the reliability and / or accuracy of theoretical models as described above, and to improve the reliability and accuracy of theoretical models. . In one particularly preferred embodiment, the method of the invention is used to characterize a plurality of different probes, eg, each probe for a distinct set of target polynucleotides, such as a distinct set of different genes or gene transcripts. A database can be constructed for properties such as specificity and sensitivity. Such databases include theoretical models of polynucleotide performance (
It is very suitable for testing and improving (for example, predictive models of polynucleotide sensitivity and specificity). Such theoretical models include those described in US Provisional Application No. 60 / 144,382 filed on July 16, 1999 and US Patent No. 09 / 364,751 filed on July 30, 1999. Models are listed.

6. Examples The following examples of assessing different probe molecules are illustrative of the methods and compositions of the invention described above and are not meant to limit the invention in any way. Specifically, the examples presented herein describe the selection of multiple oligonucleotide probes and the assessment of their sensitivity and specificity for the yeast Saccharomyces cerevisiae gene YER019W. . The results described herein demonstrate that probes that are both sensitive and specific to a particular target can be readily identified using the methods of the invention described above.

YER019W is a known gene of the yeast Saccharomyces cerevisiae, approximately 1.4 kilobases long (GenBank accession number U18778) (ie slightly longer than medium length yeast ORFs). Although the function of this gene is unknown, it has the property of being an excellent test candidate for probe selection by the method of the present invention. First, the sequence of this gene is unique and the usual BLAST comparison of that sequence to that of the yeast genome (Alts
chul et al., 1990, J. Mol. Biol. 215: 403-410; Altschul, 1997, Nucleic Acids.
Res. 25: 3389-3402; and Zhang and Madden, 1997, Genome Res. 7: 649-656.
) Does not show close homologues. This property makes YER019W suitable for testing partial specificity for overall cross-hybridization. Furthermore, YER019W is expressed at very low levels in wild type cells (1
(About 1 copy per cell). Thus, detection of wild-type expression levels by hybridization requires highly sensitive probes, but their abundance in wild-type cells is not undetectably low.

For the evaluation, the oligonucleotide probe was used as a base sequence of YER019W (GenBank
Sequences complementary to accession number U18778) were identified and selected every 25 mer. Thus, the candidate probe is a YER0 complementary to bases 1-25, 3-27, 5-29, etc.
The entire 19W sequence consisted of 705 25-mer sequences in total.

Three sets of control oligonucleotide probes were also selected. The first set consists of 50 25mer probes complementary to the yeast gene YGR192C (GenBank Accession No. Z72977), a housekeeping gene highly expressed in yeast (about 200-400 copies per cell). It was. Thus, the probe obtained from this gene can be used as a positive control for labeling and non-specific hybridization. This is because the signal intensity obtained from hybridization to this probe should always be high. The second set of control probes is the yeast gene YLR040C (GenBank Accession No. Z732), a gene of unknown function that is expressed at very low levels in yeast (less than 1 copy per cell).
It consisted of 200 kinds of 25mer probes complementary to 12). These probes were then used as a positive control for sensitivity. Control probes for both the first and second sets were also gene YGR192C and YLR040C.
In each randomly selected part, the parts were selected so that they were lined up without a gap (tiling).

The third set of probes is a yeast deletion consortium barcode (Shoemaker) so that the third set of probes are random nucleotide sequences that are maximally orthogonal to each other and are not closely related to any of the native sequences in yeast. Et al., 1996, Nature Genetics 14: 450-456.
) Was selected from 43 types of 20-mer sequences. These probes were then used as negative controls for yeast sequence hybridization.

All selected YER019W oligonucleotides, YGR192C and YLR040C controls and negative controls were tested using the standard inkjet printing method of Blanchard (International Publication WO98 / 41531 published September 24, 1998; Blanchard et al., 1996, Biosensors and Bioelectro
nics 11: 687-690; Blanchard, 1998, "Synthetic DNA arrays in genetic engineering" (Sy
nthetic DNA Array in Genetic Engineering), Volume 20, JK Setlow (ed.), Ple
num Press, New York, pp. 111-123) and double printed on the top and bottom halves of three chips and designated chips 978, 979 and 1136. All chips
200 μL of hybridization solution (10 mM Tris pH 7.6,1 M Na
Cl, 1% Triton-X-100; 1 μg / μL bovine serum albumin, 0.1 μg / μL sheared herring sperm DNA, 50 pM Cy3-labeled gridline oligonucleotide, and 50
(consisting of pM Cy5-labeled grid line oligonucleotide). After hybridization, chip at room temperature for 10 seconds, 6X SSPE, 0.005% Tri
It was shaken in ton-X-100 and further shaken in 0.06 × SSPE at room temperature for 10 seconds for washing. The chips were dried under a pressurized atmosphere and scanned with a General Scanning 3000 confocal laser scanner.

Chips 978 and 979 were simultaneously hybridized with the separately labeled samples. The first sample, consisting of 1.6 ng of fluorescently labeled fragmented YER019W cRNA, was used as the specific hybridization sample. This concentration of YER019W RNA corresponds to about 10 copies of YER019W transcript per cell, that is, about 10 times the abundance of natural YER019W. The second sample, used as a non-specific hybridization sample, was fluorescently labeled, derived from yer019w /-(ie, a diploid yeast strain specifically deleted for the gene YER019W) homozygous fission yeast mRNA. It consisted of 2 μg of fragmented cRNA. Specifically, chip 978 contains Cy5-labeled YER019W cRNA and Cy3.
Hybridized with labeled yer019w / -cRNA. Chip 979 hybridized with Cy3-labeled YER019W cRNA and Cy5-labeled yer019w / -cRNA. Chip 1136 contains 2 µg of Cy5-labeled fragmented cRNA derived from wild-type yeast and 2 µg of Cy3-labeled fragmented cRNA derived from yerO19w /-.
Hybridized with RNA.

The composite color images of the three types of chips 978, 979 and 1136 are shown in FIGS. Each of the two hybridization samples is distinguishable by the different fluorescent colors of their respective labels: "green" fluorescent Cy3 (Figs. 2A, 3A and 4A) and "red" fluorescent Cy5 (. 2B, 3B and 4B).

The gene-specific signal obtained from these images (ie the specific hybridization sample, obtained from YER019W) was synthesized from chips 978 and 979. Figure 5A shows a plot of gene tiling position versus synthesized signal.
Shown in. Similarly, non-specific signals (i.e. non-specific hybridization samples, obtained from yerO19w /-) were similarly synthesized from chips 978 and 979,
The tiling position of the gene relative to the signal is plotted in Figure 5B.

The signal shown in FIG. 5A is an indicator of the sensitivity of each probe derived from the gene YER019W. Specifically, the peaks in the plot shown in Figure 5A are
Probes that hybridize well (ie are sensitive) to YER019W and therefore may be preferred probes for detecting the gene in a polynucleotide sample are shown. The signal shown in Figure 5B indicates the amount of cross-hybridization. The peaks in this plot indicate probes that cross-hybridize with other polynucleotide sequences, which include a particularly large number of different polynucleotide sequences for these probes to specifically detect the YER019W sequence. It is suggested that it would be unfavorable for specific detection in a sample (eg a sample consisting of a large number of different yeast polynucleotide sequences extracted from cells). FIG. 5C shows a plot of the ratio of the gene-specific (ie, YER019W) signal in FIG. 5A to the gene-nonspecific (ie, yer019w / −) signal in FIG. 5B.
Therefore, this plot shows the specificity of each probe. The peak of this plot is YE
A probe that hybridizes well to R019W, while at the same time showing only limited cross-hybridization to other polynucleotides is shown. The data in Figures 5A and 5C also show that sensitivity and specificity can generally be well correlated.

FIG. 6 shows the relationship between sensitivity (GS signal, horizontal axis) and specificity (GS / GNS signal, vertical axis) for each complementary probe of YER019W using the data of FIGS. 5A and 5C. The scatter plot shown is shown. Probes exhibiting both high sensitivity and specificity (i.e., upper right of the scatter plot) are used, for example, in microarrays to detect the YER019W gene in samples of many different genes and / or gene transcripts. Is a particularly preferable probe. Such a preferred probe is also indicated by (x) in Figure 5C.

Thus, the methods and compositions described above allow for selection of the most specific and sensitive probes for detecting a particular polynucleotide (such as a particular gene).

7. Citations All references cited herein are to the same extent as if each individual publication or patent publication or patent application specification is specifically and individually indicated. As in their entirety by reference, they are incorporated by reference in their entireties in all senses.

It will be apparent to those skilled in the art that numerous modifications and variations of the present invention can be made without departing from the spirit and scope of the invention. The specific embodiments described herein are provided by way of example only, and the invention is intended to include the full scope of equivalents to which the appended claims apply. Limited only by

[Brief description of drawings]

1 is used to evaluate oligonucleotide probes on a microarray,
3 shows a flow chart illustrating an exemplary embodiment of the method of the invention.

FIGS. 2A-2B show the fluorescence obtained from the co-hybridization of two samples into a microarray containing an oligonucleotide probe complementary to the yeast Saccharomyces cerevisiae gene YER019W and the control oligonucleotide probe shown. An intensity image is shown. FIG. 2A shows a Cy3 labeled yer019w /-homozygous disrupted yeast-derived fragmented cRNA 2μ.
Fluorescence intensity of sample in g (green channel). FIG. 2B is the fluorescence intensity of the fragmented pure YER019W 1.6 ng sample labeled with Cy5 (red channel).

3A-3B show fluorescence intensity images obtained from simultaneous hybridization of two samples to the same microarray as the array of FIGS. 2A-2B. FIG. 3A is the fluorescence intensity of the fragmented pure YER019W 1.6 ng sample labeled with Cy3 (green channel). FIG. 3B shows 2 μ of fragmented cRNA derived from yer019w /-homozygous disrupted yeast labeled with Cy5.
Fluorescence intensity of sample in g (red channel).

4A-4B show fluorescence intensity images obtained from simultaneous hybridization of two samples onto the same microarray as the array of FIGS. 2A-2B. FIG. 4A shows the fragmented cRNA from Cy3 labeled yer019w /-homozygous disrupted yeast 2μ.
Fluorescence intensity of sample in g (green channel). FIG. 4B is the fluorescence intensity of a 2 μg sample of fragmented cRNA derived from wild-type yeast labeled with Cy5 (red channel).

Figures 5A-C show the amount of target and non-target hybridization observed with individual probes plotted according to their "tiling position". FIG. 5A is a plot of average normalized hybridization intensities from the combination of signals of hybridization of labeled YER019W in FIGS. 2B and 3A. FIG. 5B is a plot of average normalized hybridization intensities from the signal combinations of hybridization of labeled yer019w / − fragmented cRNA in FIGS. 2A and 3B. FIG. 5C is a plot of ratio of target hybridization intensity to non-target hybridization intensity plotted in FIGS. 5A and 5B, respectively.

FIG. 6: Scattering showing the relationship between sensitivity (“GS signal”, horizontal axis) and specificity (“GNS signal”, vertical axis) of YER019W to each complementary probe using the data in FIGS. 5A and 5C, respectively. It is a plot.

FIG. 7 shows an example of a computer system that can be used to carry out the analysis method of the present invention.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F term (reference) 2G045 AA35 DA12 DA13 DA14 FB02                       FB07 FB12 HA09 JA01                 4B024 AA11 AA19 BA80 CA04 CA05                       CA09 CA10 CA12 HA08 HA11                       HA12                 4B063 QA01 QA08 QA19 QQ01 QQ28                       QQ34 QQ40 QQ41 QQ42 QQ49                       QQ52 QQ53 QR08 QR14 QR20                       QR31 QR32 QR36 QR42 QR50                       QR55 QR56 QR58 QR59 QR62                       QR66 QS25 QS32 QS34 QS39                       QX02

Claims (89)

[Claims] 1. A method for evaluating the binding of a probe to a target molecule, comprising:
Comparing the amount of binding of molecules in the first sample to the probe with the amount of binding of molecules in the second sample to the probe; (a) the first sample comprises a plurality of identical target molecules; (B) The second sample contains a plurality of different target molecules. 2. The method of claim 1, wherein the first sample is a substantially pure sample of the same target molecule. 3. The method of claim 2, wherein the first sample is at least 75% pure. 4. The method of claim 2, wherein the first sample is at least 90% pure. 5. The method of claim 2, wherein the first sample is at least 95% pure. 6. The method of claim 2, wherein the first sample is at least 99% pure. 7. The method of claim 1, wherein each different target molecule in the second sample is different than the same target molecule in the first sample. 8. The method of claim 1, wherein the sensitivity of the probe is determined. 9. The method according to claim 8, wherein the sensitivity of the probe is determined from the amount of the specific target molecule bound to the probe in the first sample. 10. The method of claim 1, wherein the specificity of the probe is determined. 11. The specificity of the probe is determined from the ratio of the amount of the same target molecule bound to the probe in the first sample to the amount of different target molecule bound to the probe in the second sample. Item 11. The method according to Item 10. 12. The method of claim 1, wherein the same target molecule in the first sample is detectably labeled. 13. The method of claim 1, wherein the different target molecules in the second sample are detectably labeled. 14. The molecule of claim 12, wherein the molecule is detectably labeled with a fluorescent molecule.
Or the method described in 13 above. 15. (a) The same target molecule in a first sample is detectably labeled with a first label, and (b) a different target molecule in a second sample with a second label. 2. The method of claim 1, wherein the method is detectably labeled and the first label is distinguishable from the second label. 16. The method of claim 15, wherein the first label is a first fluorescent molecule and the second label is a second fluorescent molecule. 17. The method of claim 1, wherein the probe is bound to the support surface. 18. The method of claim 1, wherein the probe is one of a plurality of probes. 19. The plurality of probes comprises an array of probes, the array having a support having at least one surface, each probe bound to a support surface at a different location on the surface. Item 18. The method according to Item 18. 20. The method according to claim 1, wherein the probe is a polynucleotide probe having a specific nucleotide sequence. 21. The method of claim 20, wherein the same target molecule in the first sample is a polynucleotide molecule. 22. A specific nucleotide sequence of a polynucleotide probe is
22. The method of claim 21, which is complementary to at least a portion of the nucleotide sequence of the polynucleotide molecule in the first sample. 23. The method of claim 21, wherein the different target molecule in the second sample is a polynucleotide molecule having a polynucleotide sequence that differs from the nucleotide sequence of the polynucleotide molecule in the first sample. 24. A polynucleotide probe is bound to the surface of a support,
21. The method of claim 20. 25. The method of claim 20, wherein the polynucleotide probe is one of a plurality of polynucleotide probes having different nucleotide sequences. 26. A plurality of polynucleotide probes comprises an array of polynucleotide probes, the array having a support having at least one surface, each polynucleotide probe on a support surface at a different location on the surface. 26. The method of claim 25, which is bound. 27. A method for evaluating the binding of a polynucleotide probe having a specific nucleotide sequence to a target polynucleotide, wherein the hybridization amount of the polynucleotide in the first sample with the polynucleotide probe is Comparing the amount of hybridization of the polynucleotide in the sample with a polynucleotide probe, (a) the first sample comprises a plurality of identical target polynucleotides having a target nucleotide sequence, and (b) a second sample. Wherein the method comprises a plurality of different polynucleotide molecules each having a different nucleotide sequence. 28. A specific nucleotide sequence of a polynucleotide probe comprises:
28. The method of claim 27, which is complementary to at least a portion of the target nucleotide sequence of the target polynucleotide in the first sample. 29. The method of claim 27, wherein the target polynucleotide in the first sample corresponds to a cell or organism gene or gene transcript, or an mRNA, cDNA or cRNA derived therefrom. 30. The method of claim 27, wherein the different polynucleotide molecules in the second sample correspond to different genes or gene transcripts of a cell or organism. 31. The method of claim 27, wherein the first sample is a substantially pure sample of the target polynucleotide molecule. 32. The first sample is at least 75% pure.
The method according to 1. 33. The first sample is at least 90% pure.
The method according to 1. 34. The first sample is at least 95% pure.
The method according to 1. 35. The method of claim 3, wherein the first sample is at least 99% pure.
The method according to 1. 36. The method of claim 31, wherein each different polynucleotide molecule in the second sample has a nucleotide sequence that differs from the target nucleotide sequence. 37. (a) The target polynucleotide in the first sample corresponds to a gene or gene transcript of a cell or organism, and (b) the second sample is derived from a deletion mutant of the cell or organism. 4. A polynucleotide sample comprising a deletion mutant of said cell or organism, characterized in that it does not express the gene or gene transcript corresponding to the target polynucleotide in the first sample.
The method according to 6. 38. The plurality of different polynucleotide molecules in the second sample are (a) a polynucleotide molecule having the same nucleotide sequence as the target nucleotide sequence in the first sample, and (b) different from the target nucleotide sequence. 32. The method of claim 31, comprising a plurality of different polynucleotide molecules, each having a different nucleotide sequence. 39. (a) the target polynucleotide corresponds to a gene or gene transcript of a cell or organism, and (b) the second sample comprises a polynucleotide sample from a wild-type strain of the cell or organism, 39. The method according to claim 38, characterized in that the wild type strain of the cell or organism expresses the gene or gene transcript corresponding to the target polynucleotide. 40. (a) a first sample further comprises a polynucleotide molecule having a nucleotide sequence different from the target nucleotide sequence of said same target polynucleotide, and (b) a second sample comprises said same target polynucleotide. 28. The method of claim 27, wherein the method is absent. 41. The method of claim 40, wherein each different polynucleotide molecule in the second sample has a nucleotide sequence that differs from the target nucleotide sequence. 42. (a) the target polynucleotide corresponds to a gene or gene transcript of a cell or organism, and (b) the first sample expresses the gene or gene transcript corresponding to the target polynucleotide, or A polynucleotide sample from a wild-type strain of an organism, (c) wherein the second sample does not express the gene or gene transcript corresponding to the target polynucleotide; 42. The method of claim 41, comprising: 43. (a) the first sample further comprises a polynucleotide molecule having a nucleotide sequence different from the target nucleotide sequence of the same target polynucleotide, (b) the second sample comprises (i) the target nucleotide sequence. A polynucleotide molecule having the same nucleotide sequence as described above, and (ii) a plurality of different polynucleotide molecules each having a different nucleotide sequence different from the target nucleotide sequence, the polynucleotide molecule in the first sample having the target nucleotide sequence 28. The method of claim 27, characterized in that the amount differs from the amount of polynucleotide molecule in the second sample having the target nucleotide sequence by at least 2-fold. 44. The method of claim 43, wherein the amount of polynucleotide molecules in the first sample having the target nucleotide sequence differs from the amount of polynucleotide molecules in the second sample having the target nucleotide sequence by at least 4-fold. the method of. 45. The method of claim 43, wherein the amount of polynucleotide molecules in the first sample having the target nucleotide sequence differs from the amount of polynucleotide molecules in the second sample having the target nucleotide sequence by at least 8-fold. the method of. 46. The method of claim 43, wherein the amount of polynucleotide molecules in the first sample having the target nucleotide sequence differs from the amount of polynucleotide molecules in the second sample having the target nucleotide sequence by at least 20-fold. the method of. 47. The method of claim 43, wherein the amount of polynucleotide molecules in the first sample having the target nucleotide sequence differs from the amount of polynucleotide molecules in the second sample having the target nucleotide sequence by at least 100-fold. the method of. 48. The amount of each different polynucleotide molecule in the different molecules of the first sample is up to 100 times less than the amount of the corresponding different polynucleotide molecule in the different polynucleotide molecules of the second sample. Depends on the difference of
The method of claim 43. 49. The amount of each different polynucleotide molecule in the plurality of different molecules of the first sample is less than 10 times the amount of the corresponding different polynucleotide molecule in the plurality of different polynucleotide molecules of the second sample. 44. The method of claim 43, differing by the difference between. 50. The amount of each different polynucleotide molecule in the plurality of different molecules of the first sample is less than or equal to 50% of the amount of the corresponding different polynucleotide molecule in the plurality of different polynucleotide molecules of the second sample. 44. The method of claim 43, differing by the difference between. 51. The average amount of different polynucleotide molecules in the different polynucleotide molecules of the first sample is the same as the average amount of different polynucleotide molecules in the different polynucleotide molecules of the second sample. 44. The method of claim 43, which differs by no more than a factor of two. 52. The average amount of different polynucleotide molecules in the different polynucleotide molecules of the first sample is the same as the average amount of different polynucleotide molecules in the different polynucleotide molecules of the second sample. 44. The method of claim 43, which differs by no more than 50%. 53. The average amount of different polynucleotide molecules in the different polynucleotide molecules of the first sample is the same as the average amount of different polynucleotide molecules in the different polynucleotide molecules of the second sample. 44. The method of claim 43, which differs by no more than 10%. 54. The average amount of different polynucleotide molecules in the different polynucleotide molecules of the first sample is the same as the average amount of different polynucleotide molecules in the different polynucleotide molecules of the second sample. 44. The method of claim 43, which differs by no more than 1%. 55. The method of claim 2, wherein the sensitivity of the polynucleotide probe is determined.
7. The method according to 7. 56. The method of claim 55, wherein the sensitivity of the polynucleotide probe is determined from the amount of hybridization of the target polynucleotide molecule with the polynucleotide probe in the first sample. 57. The method of claim 2, wherein the specificity of the polynucleotide probe is determined.
7. The method according to 7. 58. The specificity of the polynucleotide probe is determined by increasing the hybridization amount of the target polynucleotide molecule in the first sample with the polynucleotide probe to the polynucleotide probe of the polynucleotide molecule in the second sample. 58. The method according to claim 57, which is determined from the ratio to the amount of hybridization. 59. The method of claim 27, wherein the target polynucleotide molecule in the first sample is detectably labeled. 60. The method of claim 27, wherein the polynucleotide molecule in the second sample is detectably labeled. 61. The method of claim 59 or 60, wherein the polynucleotide molecule is labeled with a fluorescent molecule. 62. (a) a target polynucleotide molecule in a first sample is labeled with a first label; (b) a polynucleotide molecule in a second sample is labeled with a second label; 28. The method of claim 27, wherein the label of the is distinguishable from the second label. 63. The method of claim 62, wherein the first label is a first fluorescent molecule and the second label is a second fluorescent molecule. 64. A polynucleotide probe is bound to the surface of a support,
The method of claim 27. 65. The method of claim 27, wherein the polynucleotide probe is one of a plurality of polynucleotide probes. 66. A plurality of polynucleotide probes comprises an array of polynucleotide probes, the array having a support having at least one surface, each probe bound to a support surface at a different location on the surface. 66. The method of claim 65, characterized in that 67. A method of assessing the binding of a plurality of polynucleotide probes to a target polynucleotide, wherein each polynucleotide probe in the plurality of polynucleotide probes has a specific nucleotide sequence, the method comprising: The amount of hybridization between the polynucleotide in the first sample and each of the polynucleotide probes in the plurality of polynucleotide probes is calculated by comparing the amount of hybridization between the polynucleotide in the second sample and each of the polynucleotide probes in the plurality of polynucleotide probes. (A) the first sample comprises a plurality of the same target polynucleotides having a target nucleotide sequence, and (b) the second sample comprises a plurality of different nucleotide sequences each having a different nucleotide sequence. Contains polynucleotide molecules The above method, characterized in that 68. The method of claim 67, wherein the particular nucleotide sequence of each polynucleotide probe is complementary to at least a portion of the target nucleotide sequence of the target polynucleotide in the first sample. 69. The method of claim 67, wherein the sensitivity of each polynucleotide probe in the plurality of different polynucleotide probes is determined. 70. The sensitivity of each polynucleotide probe in a plurality of different polynucleotide probes is determined by the amount of hybridization between the same target polynucleotide molecule in a first sample and each polynucleotide probe in the plurality of polynucleotide probes. 70. The method of claim 69, determined from 71. The method of claim 69, wherein the specificity of each polynucleotide probe in the plurality of different polynucleotide probes is determined. 72. The specificity of each polynucleotide probe in the plurality of polynucleotide probes is determined by: (a) the amount of hybridization of the same target polynucleotide molecule in the first sample with each polynucleotide probe; 72. The method of claim 71, which is determined from the ratio of a plurality of different polynucleotide molecules in a second sample to the amount of hybridization with each polynucleotide probe. 73. The method of claim 67, wherein each polynucleotide probe in the plurality of polynucleotide probes is bound to the support surface. 74. A plurality of polynucleotide probes comprises an array of probes, the array having a support having at least one surface, each probe in the plurality of probes at a different location on the surface. 68. The method of claim 67, wherein the method is associated with 75. The first sample comprises two or more different target polynucleotide molecules, each of said two or more different target polynucleotide molecules being said
68. The method of claim 67, wherein the method does not hybridize or cross-hybridize with a probe that also hybridizes or cross-hybridizes with another species of one or more different target polynucleotide molecules. 76. A method of assessing the hybridization conditions of one or more polynucleotide probes, wherein said one or more polynucleotide probes each have a particular nucleotide sequence, said method comprising: Under specific hybridization conditions, the hybridization amount of the polynucleotide in the first sample with each of the one or more polynucleotide probes is determined by the amount of the one or more polynucleotides in the second sample. Comparing the amount of hybridization with each of the above polynucleotide probes, (a) the first sample contains a plurality of the same target polynucleotides having a target nucleotide sequence, and (b) the second sample respectively. Have different nucleotide sequences Comprising a plurality of different polynucleotide molecules, characterized in that, the method described above. 77. Determining the sensitivity of each of one or more polynucleotide probes under specific hybridization conditions.
The method according to 6. 78. Sensitivity of each of one or more polynucleotide probes to a plurality of same target polynucleotide molecules and one or more polynucleotides in a first sample under specific hybridization conditions. 8. Determined from the amount of hybridization with each of the probes.
7. The method according to 7. 79. The specificity of each of the one or more polynucleotide probes is determined under specific hybridization conditions.
The method according to 6. 80. The specificity of each of the one or more polynucleotide probes is determined by: (a) a plurality of the same target polynucleotide molecules in the first sample under one or more specific hybridization conditions; (B) a plurality of different polynucleotide molecules in the second sample and each of one or more polynucleotide probes in the hybridization amount with each of the above-mentioned polynucleotide probes, 80. The method according to claim 79, which is determined from the ratio of to the amount of hybridization. 81. Prior to the step of comparing above, further comprising the following steps: (i) contacting the probe with the first sample under conditions conducive to binding, and (ii) conditions contributing to binding. Contacting the probe with a second sample below, (iii) detecting the bond between the probe and the molecule in the first sample, and (iv) detecting the bond between the probe and the molecule in the second sample. The method of claim 1, comprising: 82. The method of claim 8 wherein said contacting steps (i) and (ii) are performed simultaneously.
The method according to 1. 83. The method according to claim 82, wherein said detecting steps (iii) and (iv) are performed simultaneously. 84. The polynucleotide in the first sample is labeled with a first label and the polynucleotide in the second sample is labeled with a second label distinguishable from the first label, the comparison as above. Prior to the step of: (i) contacting the polynucleotide probe with the first sample and the second sample simultaneously under conditions that contribute to hybridization, (ii) the polynucleotide probe and the first sample; And detecting the binding that occurs between the polynucleotide in the second sample and the polynucleotide in the second sample. 85. The method of any one of claims 81-84, wherein the second sample does not contain the same target molecule or polynucleotide in the first sample. 86. A computer system for assessing the binding of a probe to a target molecule comprising a memory and a processor element interconnected with the memory, the memory comprising: Encoding one or more programs, the method comprising: comparing the amount of a molecule in the first sample bound to the probe to the amount of a molecule bound to the probe in a second sample; ) A system as described above, characterized in that the first sample comprises a plurality of identical target molecules, and (b) the second sample comprises a plurality of different target molecules. 87. A computer system for assessing the binding of a polynucleotide probe, comprising: a memory; and a processor element interconnected with the memory, the memory having the processor element have the following method: One or more programs for carrying out a method comprising: comparing the amount of hybridization of a polynucleotide in a sample to a polynucleotide probe to the amount of hybridization of a polynucleotide in a second sample to a polynucleotide probe. (A) a first sample comprises a plurality of identical target polynucleotides having a target nucleotide sequence, and (b) a second sample comprises a plurality of different polynucleotide molecules each having a different nucleotide sequence, Featuring That, the above-mentioned system. 88. A computer program product for use with a computer having a memory and a processor element, including a computer readable recording medium having a coded computer program mechanism, the computer program mechanism in the memory. The processor element may be loaded and comprises the following method: Comparing the amount of molecules bound to the probe in the first sample with the amount of molecules bound to the probe in the second sample. , (A) the first sample comprises a plurality of the same target molecules, and (b) the second sample comprises a plurality of different target molecules. The above computer program product. 89. A computer program product for use with a computer having a memory and a processor element, including a computer readable recording medium having a coded computer program mechanism, the computer program mechanism in the memory. The processor element may be loaded and the method is as follows: hybridization amount of a polynucleotide in a first sample to a polynucleotide probe and hybridization of a polynucleotide in a second sample to a polynucleotide probe. Encoding one or more programs for carrying out the method, comprising: (a) a first sample containing a plurality of identical targets having a target nucleotide sequence; Include Li nucleotides, (b) the second sample comprises a plurality of different polynucleotide molecules having different nucleotide sequences, respectively, characterized in that, the computer program product.