JP2001511360A - Multifunctionality and its use within array elements - Google Patents

Abstract

  (57) [Summary] The present invention provides an array of oligonucleotides on a solid substrate, wherein distinct regions have at least two oligonucleotides with different sequences. These arrays are useful in hybridization assays, particularly in conjunction with cleavable mass spectrometry tags.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates generally to solid substrates having an array of oligonucleotides printed on a surface, and more particularly to an array having a plurality of oligonucleotides of various sequences in discrete regions of the array. .

BACKGROUND OF THE INVENTION [0002] Replicated arrays of biological agents have been used to facilitate parallel testing of many samples. For example, as a means of rapidly screening many independent colonies for different growth phenotypes, bacterial and yeast colonies were used on agar plates, each containing a different growth medium, using sterile velvet cloth and piston ring devices. Have been produced (Lederberg and Lede
rberg, J .; Bacteriol. 63: 399,1952). Similarly, 9
Using a 6-well microtiter plate, a number of, for example, cell lines, virus isolates representing recombinant DNA libraries, or monoclonal antibody cell lines are organized and stored in an easily accessible manner.

[0003] The advent of large-scale genome projects and the increasing use of molecular diagnostics has required the development of high-throughput methods for screening nucleic acids. Recently, methods have been developed to synthesize large arrays of short oligodeoxynucleotides (ODNs) attached to a glass or silicon surface that represent all or a subset of all possible nucleotide sequences (Maskos and Southern, Nucl. Aci
ds Res. 20: 1675, 1992). These ODN arrays were hybridized (Southern et al., Genomics 13: 1008,
1992; Drmanac et al., Science 260: 1649, 1993).
Has been used to perform DNA sequence analysis, determine the expression profile, and screen for mutations. For all of these uses, the number of oligonucleotides required is large, and high-density arrays (> 1000 oligonucleotides per cm ² ) have been developed. However, using lower density arrays is advantageous in terms of time and is economical. Currently, such arrays are limited by attaching the oligonucleotide (often in situ synthesis) and the various detectable markers.

[0004] The present invention discloses methods and compositions for producing arrays having more than one nucleotide sequence per distinct region, and further provides other related advantages.

SUMMARY OF THE INVENTION In one aspect of the invention, an array of oligonucleotides comprising a solid substrate having a surface containing discrete regions of nucleic acid molecules, preferably oligonucleotides, wherein at least one of the oligonucleotides comprises An array is provided wherein at least two regions comprise at least two nucleic acid molecules selected to have different sequences. Preferably, 1000
There are less distinct areas. Also preferably, each region comprises at least two oligonucleotides having different sequences, and more preferably, at least one region comprises from 2 to about 100 different oligonucleotide sequences.

[0006] For certain embodiments, the areas of the array are about 20 to about 500 microns in diameter and the center-to-center distance between the areas is about 50 to 1500 microns.

In a preferred embodiment, the oligonucleotide has a known sequence.
In another preferred embodiment, the oligonucleotide is preferably a poly (
It is covalently attached to the surface of the substrate via an amine bond such as ethyleneimine).

[0008] In another aspect, the present invention relates to a method for hybridization analysis,
The following steps: (a) hybridizing a labeled nucleic acid molecule to the array of oligonucleotides of claim 1; and (b) detecting a label in a region of the array, thereby identifying any label on the array. Determining whether the oligonucleotide has hybridized. In a preferred embodiment, at least one region of the array comprises an oligonucleotide of known sequence, and one of the labeled nucleic acid molecules is complementary to the oligonucleotide. Preferably, the labeled nucleic acid molecules include nucleic acid molecules having different sequences, each with a different label. Such labels may be selected from the group of radioactive molecules, fluorescent molecules, and cleavable mass spectrometry tags.

In another aspect, the present invention is a method for identifying a nucleic acid molecule in a sample, comprising the following step (a): hybridizing a labeled oligonucleotide to the nucleic acid molecule;
Forming a duplex; (b) isolating the duplex; (c) denaturing the duplex; (d) labeling the labeled oligonucleotide as described herein. Hybridizing to an array of oligonucleotides, wherein the oligonucleotides on the array are complementary to the labeled oligonucleotides; and (e) detecting a label in a region of the array. Identifying a nucleic acid molecule in the sample, thereby providing a method comprising:

In yet another aspect, a method for identifying a nucleic acid molecule in a sample, the method comprising: (a) hybridizing an oligonucleotide to the nucleic acid molecule; (b)
Elongating the oligonucleotide in the presence of a single labeled nucleotide to form a duplex; (c) denaturing the duplex; (d) modifying the labeled oligonucleotide herein. Hybridizing to an array of oligonucleotides as described herein, wherein the oligonucleotides on the array are complementary to the labeled oligonucleotides; and (e) labeling within a region of the array. And thereby identifying the nucleic acid molecule in the sample. In a preferred embodiment, the oligonucleotides in discrete regions of the array are complementary to the extension product of the nucleic acid molecule.

In yet another aspect, the present invention is a method for identifying a nucleic acid molecule in a sample, comprising the following step (a) wherein at least two oligonucleotides are hybridized to the nucleic acid molecule, Wherein at least one oligonucleotide is labeled, and the oligonucleotide hybridizes to an adjacent sequence on the nucleic acid molecule; (b) joining the oligonucleotide; (C) denaturing the duplex; (d) hybridizing the labeled oligonucleotide to an array of oligonucleotides described herein, wherein the oligonucleotides on the array are Is complementary to the ligated oligonucleotide; and A method that does not occur in nucleotides; and (e) detecting a label in a region of the array; thereby identifying a nucleic acid molecule in the sample.

In yet another aspect, a method for identifying an mRNA molecule in a sample, comprising the following step (a): hybridizing a labeled oligonucleotide to the mRNA molecule to form a duplex. (B) isolating the duplex; (c) denaturing the duplex; (d) hybridizing the labeled oligonucleotide to an array of oligonucleotides as described herein. Wherein the oligonucleotides on the array are complementary to the labeled oligonucleotides; and (e) detecting a label in a region of the array; thereby, detecting the mRNA in the sample. Identifying the molecule. In a preferred embodiment, the mRNA molecule is isolated from cells that have been treated with the suspected toxic compound. In another preferred embodiment, the oligonucleotides on the array are sequences of a cytokine.

[0013] These and other aspects of the invention will become apparent with reference to the following detailed description and accompanying drawings. Additionally, various references are set forth below, which describe particular procedures or compositions in more detail (eg, plasmids, etc.) and are therefore incorporated by reference in their entirety.

The biomolecule arrays of the present invention can contain or be used with tagged biomolecules, eg, oligonucleotides covalently linked to a cleavable tag. These tagged biomolecules can be used in the methods of the invention, and in assay procedures such as oligonucleotide sequencing and gene expression assays, among others. Exemplary tagged biomolecules, and assays that can use this same, are described in U.S. patent application Ser. Nos. 08 / 786,835, filed Jan. 22, 1997;
Nos. 86,834; and 08 / 787,521; and 08 / 98,180, each filed on July 22, 1997;
No. 64; and three U.S. Patents Nos. 08 / 98,501; and PCT International Publication No. WO 97/27331;
No. 27325; and WO 97/27327. Each of these six U.S. patent applications and three PCT publications is incorporated herein by reference in its entirety.

The biomolecule array of the present invention is also disclosed in “Amp,” filed on Jul. 22, 1997.
lifcation And Other Enzymatic React
US Provisional Patent Application No. 60 / 053,428, entitled "Ions Performed On Nucleic Arrays," and co-filed similarly non-provisional US Non-Provisional Patent Application No. (), both of which are incorporated herein in their entirety. As described in US Pat.

The biomolecule array of the present invention, and the array useful for the method of the present invention include, for example, 19
"Apparatus And Method, filed July 22, 1997
US Provisional Patent Application No. 60 / 053,435, entitled "For Arraying Solution On A Solid Support," and co-filed similarly-provisioned U.S. Non Provisional Application No. (), both of which are incorporated herein by reference. Which are incorporated by reference herein in their entirety).

The biomolecule array of the present invention and the array useful for the method of the present invention include, for example, 19
"Polyethylenimine-Bas" filed on July 22, 1997
US Provisional Patent Application No. 6 entitled "ed Biomolecule Arrays"
No. 0 / 053,352, and co-filed, similarly-titled US Provisional Application No. (), both of which are incorporated herein by reference in their entirety. Can be prepared according to

For example, “Computer Metho,” filed on July 22, 1997
US Provisional Patent Application Ser. No. 60 / 053,429, entitled "d and System for Correlating Data," and co-filed similarly non-provisional US Non-Provisional Patent Application No. (), both of which are incorporated in their entirety. Computer systems and methods of correlating data, such as those disclosed in U.S. Pat.

DETAILED DESCRIPTION OF THE INVENTION As described above, the present invention provides multiple arrays in an array in a single, discrete area. In certain embodiments, the invention provides more than one, and preferably 10-100, different oligonucleotide or polynucleotide sequences in a single element in the array. In the case of oligonucleotides, between 2 and about 100 oligonucleotides are individually synthesized on a commercial synthesizer, combined and printed as a single element in discrete regions of the array. Single or double stranded nucleic acids, including DNA, RNA or both, can be bound to a solid substrate. Double-stranded molecules can be made by amplification, enzymatic digestion, and the like. Naturally, any nucleic acid molecule having a primary amine group (for attachment to polyethyleneimine) or other reactive group can be attached to the array within the present invention. Also within the present invention, the sequence of all individual nucleic acids within a particular region may be unknown.

[0020] As used herein, an array refers to a collection of oligonucleotide or polynucleotide sequences that are placed on a solid support in discrete regions. Preferably, the regions form some identifiable pattern or regular spacing. Arrays are typically composed of 2 to 1000 elements, but can also be composed of more than 1000 elements (discrete areas). Each region is separated by some distance, where neither nucleic acids nor oligonucleotides are bound or deposited. Typical area sizes are 20 to 500 microns, and typical center-to-center distances for the area range from 50 to 1500 microns.

The present invention also describes the use of multiple sequences within a single element in an array.
This method allows the use of low element arrays (ie, 10 to 400 elements, for example) for many purposes. For example, these combined sequences, low element arrays, can be used for pathogen identification, profiling decisions, toxicology tests, and the like.

I. Application of Template to Solid Substrate A. Substrate Preparation The substrates of the array are prepared from appropriate materials. The substrate is preferably rigid and has a surface that is preferably substantially flat. In some embodiments, the surface may have raised portions to indicate areas. Exemplary substrates are silicon wafers and borosilicate slides (eg, microscope glass slides), but other materials known in the art can be substituted. An example of a particularly useful solid support is a silicon wafer, which is typically used in the electronics industry in semiconductor construction. The wafer is highly polished on one side and reflective, and can be easily coated with various linkers, such as poly (ethylene imine) using silane chemistry. Wafers are commercially available from companies such as WaferNet, San Jose, CA.

[0023] Nucleic acid molecules or other biomacromolecules, such as peptides, can be synthesized, made or isolated, and applied to a solid substrate. Nucleic acids and peptides can be synthesized in an automated manner using commercially available machines. In a preferred embodiment, the molecule is deposited on a solid substrate and covalently attached to the solid substrate.

[0024] In certain embodiments, the surface of the substrate is prepared for oligonucleotides. The surface can be prepared, for example, by coating with a chemical that increases or decreases hydrophobicity, or with a chemical that allows covalent attachment of the nucleic acid molecule or other polymeric sequence. Some chemical coatings may alter the hydrophobicity and allow for covalent bonding. Hydrophobicity on solid substrates can be easily increased by silane treatments or other treatments known in the art. Chemicals that allow covalent bonding are commonly referred to as linkers. These linker molecules include functional groups that adhere to the surface of the substrate and react with biomolecules. Many such linkers are readily available. For example, a solid support
Photolabile-protected hydroxyl groups (U.S. Pat. Nos. 5,412,087; 5,571,639; 5,593,833).
No. 9), hydroxyl groups derivatized with alkoxy or aliphatic (US Pat. No. 5,436,327), or other chemicals (eg, US Pat.
No. 5,934; EP Patent EP-B1-0,373,203; U.S. Pat.
474,796; see US Pat. No. 5,202,231).

A preferred coating that reduces hydrophobicity and provides a linker is poly (ethyleneimine). Furthermore, solid substrates coated with poly (ethylene imine) (PEI) have the advantage of long shelf life stability. Coating of silicon wafers and glass slides with polymers, such as poly (ethyleneimine), can be done in-house or at Cell Associates (Houston
, Texas). Glass slides can also be coated with a reflective material or can be coated with PEI using silane chemistry. The PEI coating can be a single or double stranded oligonucleotide, a single or double stranded long DNA molecule or fragment,
Alternatively, it allows covalent attachment of any other amine-containing biomolecules to the solid support. Oligonucleotides can be covalently attached at the 5 'using a hexylamine modification, which places a primary amine at the 5' end of the oligonucleotide. The 5 'amine on the oligonucleotide can then react with a crosslinker, such that the oligonucleotide is covalently attached to the polymer coated on the solid support.

[0026] Any nucleic acid type can be covalently attached to the PEI coated surface as long as the nucleic acid contains a primary amine. Amplification product (eg, by PCR)
Can be modified to contain a primary amine by using a 5 'hexylamine conjugated primer. The amine group is allyl-dUTP (Sigma,
St. (Louis, Mo.) can be introduced into amplification products and other nucleic acid duplexes. Moreover, amines can be introduced into nucleic acids by polymerases, such as terminal transferases, or by ligation of short amine-containing oligonucleotides. Other suitable methods known in the art can be substituted.

Crosslinkers suitable for amine groups are generally commercially available (see, for example, Pierce,
Rockford, IL). A representative crosslinking agent is trichlorotriazine (cyanuric chloride) (Van Ness et al., Nucleic Acids®).
es. 19: 3345-3350, 1991). Briefly, an excess of cyanuric chloride is a solution of an oligonucleotide solution (e.g., 1% of an amine) at a typical oligonucleotide concentration of 0.01-1 μg / ml, and preferably about 0.1 μg / ml.
0-1000-fold molar excess of cyanuric chloride). The reaction is buffered using a common buffer such as sodium phosphate, sodium borate, sodium carbonate, or Tris HCl in a pH range of 7.0 to 9.0. A preferred buffer is a freshly prepared 0.2 M borate N pH 8.3 to pH 8.5.
a. 10 μl of a 15 mg / ml solution of cyanuric chloride was added, and
The reaction is carried out for 2 hours and preferably for about 1 hour with constant stirring. Reaction temperatures can range from 20 to 50 ° C, with a preferred reaction temperature being 25 ° C (or ambient temperature).

When cyanuric chloride is used as a cross-linking agent, it is not necessary to remove excess cross-linking agent before printing the nucleic acid on a solid substrate. Excess cyanuric chloride in the reaction mixture does not interfere with or compete with the covalent attachment of the nucleic acid or oligonucleotide to the PEI-coated solid substrate. This is because the amine on the solid substrate is in excess of the number of cyanuric chloride molecules. In a preferred embodiment, the crosslinked oligonucleotide is not purified before the printing step.

If the nucleic acid or other amine-containing polymer is covalently attached, the activated polymer is reacted with the solid support for 1-20 hours at 20-50 ° C., preferably 1 hour at 25 ° C. Let it. The free amine on the solid support is then capped to prevent non-specific attachment of other nucleic acids. Capping is performed by adding the solid support to
0.1% to 0.2% in 0% m-pyrrole and 0.1 M Na borate.
It is achieved by reacting with 0 M succinic anhydride, preferably 1.0 M succinic anhydride, for a reaction time of 15 minutes to 4 hours, preferably 30 minutes at 25 ° C. The solid support is then 0.1 to 10.0 M Na borate (preferably pH 8 to pH 9 to pH 9) containing 0.1 to 5 M glycine (preferably 0.2 M glycine).
. 3 in a 0.1 M Na borate solution, and then washed with a detergent containing solution. This caps any dichlorotriazine that can be covalently attached to the PEI surface. Preferably, the solid support is 0.01 M
Further heating to 95 ° C. for 5 minutes in NaCl, 0.05 M EDTA and 10 mM Tris (pH 8.0) removes any non-covalently attached nucleic acids. If the double-stranded nucleic acid is printed on a solid substrate, this step will also convert (denature) the double-stranded to single-stranded form.

In the currently used array format, this array contains the lowest possible information content: each element of the array corresponds to exactly one sequence. therefore,
Each element in the array is of a known sequence, and if the element score is positive in the array (eg, hybridizes), the sequence in question is known. Essentially, there is one correspondence to one of the materials contained in the elements of the array and the information content contained within the array.

In the arrays of the present invention, additional informational content is provided by having multiple sequences for each element. In its simplest form, each region of the array contains a unique sequence and a control sequence for measuring the amount of material per element in the array. If two oligonucleotides are immobilized within a single element, one oligonucleotide can be used for quality control on the array or as a means to direct quality assurance. A “control” oligonucleotide sequence may serve as a capture site for a complementary oligonucleotide that contains or has some detectable label. "Control" oligonucleotides can also serve as internal controls for the arraying processes and methods.

In this format, at least 1
One region contains multiple nucleic acid sequences. In a preferred embodiment, each region contains multiple sequences. For some purposes, the median or percentage of the regions will have multiple sequences and the remaining percentages will have a single sequence. Similarly, when multiple sequences are present, there are at least two sequences, generally no more than 1000, and preferably 2 to 100 sequences.

In a preferred aspect, the array contains an intermediate amount of information content. For example, in one embodiment, the array includes two sequences per element. Therefore, there is a 2: 1 ratio between the information content per element and the number of elements per array.
However, there is a loss of correct sequence identity when using a single label in this format. All elements in this array are composed of two possible known sequences, and if the element score is positive in the array, the sequence in question is the possibility of two different sequences. However, positive identity can be determined by the use of multiple detection molecules. As described below, various fluorescent molecules, colored microbeads, radioactive molecules, chromogenic substrates, combinations of these markers, combinations of one of these markers with a chemical chromophore, or cleavage. The use of a possible mass spectrometry tag can provide information as to which sequences in a region of the array have hybridized.

As shown in the table below, as the number of different sequences increases in each region of the array, the number of repetitive reactions (deconvolutions) that need to be performed for unambiguous identification Is increased, or the number of different tags required is increased.

[0035]

[Table 1]

Where n preferably ranges from 1 to 100 per element in the array. The number of sequences per element was placed in a single element per array,
The number of different sequences. The "Is the sequence known?" Column indicates the ability to sequence the target (test) sample with a single label. Correspondence is the amount of information or likelihood that any given sequence is represented in a single element using an array or subarray. The deconvolution column shows the exact sequence of the target (test sample) (
This represents the number of sub-assays that need to be performed to determine which was a positive score in the assay). For example, if a hit occurs in a region containing ten different sequences, an unambiguous determination can be made by printing and hybridizing the ten different sequences separately.

[0037] Substantial and countless advantages are achieved using more than one nucleic acid sequence or more than one oligonucleotide per element in the array. For example, families of related nucleic acid sequences can be placed within a single element of the array, and thus the gene activity of any test sample can be determined by examining the pattern of hybridization across the array. In the case of toxicology testing, an array with the following elements can be constructed: Pro-inflammatory cytokine induction (IL
-1, IL-2, IL-6, IL-8), an element containing a sequence for anti-inflammatory cytokine induction (IL-4, IL-12),
An element containing a sequence for a lipid-modifying enzyme (platelet-activating factor acetyltransferase (platelet-activating factor acetylhydrolase); and TNF (TN
Elements containing sequences for Fα and TNFβ). One skilled in the art will recognize that the choice of nucleic acid sequence will depend, in part, on what is being tested.

B. Methods of Applying Nucleic Acid Molecules to Solid Substrates Oligonucleotides, nucleic acid molecules, or other biopolymers are “printed” (delivered or applied) on a solid substrate. In a preferred embodiment, the polymers are applied in a regular pattern or array.

A variety of printing methods are available for applying nucleic acids, eg, oligonucleotides or DNA fragments, in an array pattern to a solid substrate. As a general guideline, this delivery mechanism positions very small volumes of liquid (eg, nanoliters) in small areas (eg, 300 μm diameter dots) that are very close to each other (eg, less than 1 mm apart). It must be possible to Preferably, the printing technique can be automated. One such technique is inkjet printing using multiple heads. Very fine pipettors can also be used. The preferred means of printing uses spring probes as described herein.

Sample pickup, transfer, and microdroplet deposition are greatly enhanced when using a liquid transfer device with a hydrophilic surface, especially when the device is a modified spring probe. . Spring probes are made hydrophilic through the use of chemical agents that act to modify the surface of the probe, or through coating the probe with a hydrophilic substance. In a preferred method, the tip of the spring probe is 1,4-dithiothreitol,
. Immerse in a 25-200 mM solution of 1 M sodium borate for 15 minutes to 2 hours. Dithiothreitol reacts with the gold surface through a thiol-gold coordination, which essentially hydroxylates the surface and renders the surface hydrophilic.

The hydrophilic surface promotes uniform coating of the sample when the spring probe is immersed in a solution. Grooved probes, due to their hydrophilic nature,
The raised liquid is uniformly and consistently loaded on the probe surface. Solutions with viscosity enhancing chemicals such as glycerol provide in particular improved handling capabilities using hydrophilic surfaces. With these solutions, the glycerol adheres uniformly to the probe when drawn from a liquid source. When a sample is transferred from its source to the solid support, the hydrophilic surface of the probe will hold the transferred sample and randomly drop or flow the sample during transport. Prevention continues to benefit from liquid operations. When a sample with a spring probe comes into contact with a solid support, especially for a sample containing a viscosity enhancing solution, the sample is deposited on the surface of the solid support from the tip of the spring probe. The size of the spotted area is generally between 10 and
It is in the range of 200 μm, with a typical center-to-center distance of 25-500 μm.

Briefly, in a typical procedure, a solution of the nucleic acid is mixed evenly in 57% glycerol and then printed on the solid support. In the context of the present invention,
The biopolymer can be either a nucleic acid molecule or a protein molecule. When nucleic acids are used, these may be single- or double-stranded DNA, single- or double-stranded RNA, oligonucleotides, hybrid DNA-RNA molecules, or double-stranded, PNA nucleic acids having a protein backbone, etc. May be included.

(II. Reaction Components and Conditions) As described above, the present invention provides a method for hybridizing a nucleic acid on a solid substrate. As described above, can the nucleic acid be covalently attached to the surface of the substrate,
Or it can be deposited on this substrate without adhesion. Typically, the oligonucleotide is printed first and other reagents are subsequently added.

(A. Reagents, Buffers, Cofactors, etc.) Each region of the array that undergoes hybridization has an appropriate labeled nucleic acid, buffer, cofactor, etc. in addition to the template nucleic acid. Hybridization conditions are well known (Ausubel et al., Current Protocols in M.
Molecular Biology, Green Publishing, 1995; Sambrook et al., Molecular Cloning: A Laboratory Approach, Cold Spring.
(See Harbor Press, 1987), and hybridization using short pieces of nucleic acid is well described.

In a preferred embodiment, a hybotrope may be added to improve the annealing of the oligonucleotide primer to the template (US Application No. 60 / 026,6).
No. 21 (filed on Sep. 24, 1996); and 08 / 719,132 (filed on Sep. 24, 1996).
No. 08 / 933,924 (filed on Sep. 24, 1996);
No. 09 / 002,051 (filed on Dec. 31, 1997); and PCT International Patent Application No. WO 98/13527 (filed on Sep. 23, 1997).
All of which are incorporated herein by reference in their entirety)). Hypotrope, when referred to a standard salt solution (ie, 0.165 M NaCl), refers to any chemical that can increase the enthalpy of a nucleic acid duplex by 20% or more. The chemical exhibits hybotropic properties when the 18 bp oligonucleotide duplex, as a solution, 50% G + C, has a helix-coil transition (HCT) of 15 ° C. or less. HCT is the temperature difference where 80% and 20% of the duplexes are single-stranded. The temperature for annealing is then the discrimination temperature, at which temperature the hybridization reaction is performed so that the discrimination between mismatched and perfectly matched duplexes can be detected. To be selected. The temperature range satisfies the determination criteria for the discrimination temperature.

(III. Detection of Reaction Product) The reaction product can be detected by various methods. Preferably, one of the reaction components is labeled. In amplification reactions, oligonucleotide primers or nucleotides are conveniently labeled. Preferably, the primer contains a label. In single nucleotide extension assays, the added nucleotides are generally labeled; in oligonucleotide ligation assays, one or more oligonucleotides are labeled; in other synthetic reactions, either primers or nucleotides are used. Is typically labeled.

[0047] Commonly used labels include, but are not limited to, biotin, fluorescent molecules, radioactive molecules, chromogenic substances, chemiluminescent agents, and the like. Methods for biotinylation of nucleic acids are well known in the art, as are methods for introducing fluorescent and radioactive molecules into oligonucleotides and nucleotides.

When biotin is used, the biotin is conjugated to a detectable marker such as an enzyme (eg, horseradish peroxidase) or a radiolabel (eg, ³² P, ³⁵ S, ³³ P). It is detected by avidin, streptavidin and the like. Enzyme conjugates are available, for example, from Vector Laboratories (Bu
(Lingame, CA). Streptavidin binds biotin with high affinity, unbound streptavidin is washed away, and then the presence of horseradish peroxidase enzyme is precipitated using a substrate that precipitates in the presence of peroxide and a suitable buffer. Is detected. The product can be detected using a microscope equipped with a visible light source and a CCD camera (Princ
(Eton Instruments, Princeton, NJ). With such an instrument, about 10,000 μM × 10,000 μM images can be scanned at a time.

[0049] Detection methods are well known for such labels, such as fluorescent or radioactive labels. Fluorescent labels can be identified and quantified almost directly by their absorption and fluorescence emission wavelength and intensity. Microscope / camera equipment using a fluorescent light source is a convenient means for detecting fluorescent labels. The radiolabel can be visualized by standard autoradiography, phosphor imaging analysis or a CCD detector. Other detection systems are available and known in the art. For labels such as biotin, radioactive, fluorescent, the number of different reactions that can be detected at one time is limited. For example, the use of four fluorescent molecules (eg, commonly used in DNA sequencing analysis) limits the analysis to four samples at a time. Essentially, due to this limitation, when using these detector methods,
Each response must be evaluated individually.

A more advantageous detection method is to pool the sample reactants into at least one array,
Enables simultaneous analysis of products. By using tags with different molecular weights or other physical properties in each reaction, the entire set of reaction products can be collected and analyzed together. (For example, U.S. Patent Application Nos. 08 / 786,835, 08 / 786,834, 08 / 787,521 (filed on January 22, 1997, respectively) and 08 / 898,180. Nos. 08 / 898,564 and 08 / 898,501 (filed on July 22, 1997, respectively), and PCT International Publication Nos. 97/27331, 97/27325, and 97/27325. See, eg, 97/27327.) Briefly, “tag” molecules are used as labels. As used herein, "tag" refers to a chemical moiety used to uniquely identify a "molecule of interest" and, more specifically, a tag variable component, as well as any tag reaction. Any substance, tag component, and tag portion that can be bound in the closest proximity to it.

Tags useful in the present invention have several properties: (1) can be distinguished from all other tags. This distinction from other chemical moieties may be based on the chromatographic behavior of the tag (particularly after the cleavage reaction), its spectroscopic or potentiometric properties, or some combination thereof. Spectroscopic methods by which tags are usefully distinguished include mass spectrometry (MS), infrared (IR), ultraviolet (UV)
), And fluorescence; MS, IR and UV are preferred, and MS is the most preferred spectroscopic method. The current measurement method of the potential difference measurement is a preferable potential difference measurement method. (2) Tags can be detected when present at 10 ^-22 to 10 ^-6 mol. (3) The tag has a chemical handle through which the tag can be bound to the MOI that the tag is intended to uniquely identify. Combination is MOI
Directly or indirectly via a “linker” group. (4) The tag is chemically attached to all operations to which it is subjected, including binding to and cleavage from the MOI, and any manipulation of the MOI while the tag is attached. It is stable. (5) The tag does not significantly interfere with operations performed on the MOI while the tag is attached. For example, if the tag is attached to an oligonucleotide, the tag must not significantly interfere with any hybridization or enzymatic reactions (eg, amplification reactions) performed on the oligonucleotide.

A tag moiety intended to be detected by a particular spectroscopic or potentiometric method should have properties that enhance the sensitivity and specificity of the detection by that method. Typically, the tag portion has those properties by virtue of their properties being designed into a tag variable component that typically constitutes a major portion of the tag section. In the discussion that follows, use of the term “tag” may typically be considered to refer to the tag moiety (ie, the cleavage product containing the tag variable component), but to the tag variable component itself. Because it is typically part of the tag portion responsible for providing unique detectable properties. In compounds of Formula TLX, the "T" moiety contains the tag variable. If the tag variable is designed to be characterized, for example, by mass spectrometry, the T-LX “
T "portion may be referred to as T ^ms. Similarly, cleavage products from T- ^LX containing T can be referred to as ^Tms- containing moieties. The following spectroscopic and potentiometric methods may be used to characterize T ^ms -containing moiety.

Thus, within one aspect of the invention, there is provided a method for determining the identity of a nucleic acid molecule or fragment (or a method for detecting the presence of a selected nucleic acid molecule or fragment). . The method comprises the following steps: (a)
Hybridizing a tagged nucleic acid molecule from one or more selected target nucleic acid molecules, wherein the tag is correlated with a particular nucleic acid molecule and detected by non-fluorescence spectroscopy or potentiometry (B) washing the unhybridized, tagged nucleic acid; (c) cleaving the tag from the tagged molecule; and (d) removing the tag from the non-hybridized tag. Detecting by fluorescence spectroscopy or potentiometry and thereby determining the identity of the nucleic acid molecule. Examples of such techniques include, for example, mass spectrometry, infrared spectroscopy, potentiostatic amperometry or UV spectroscopy.

IV. Use As described above, the method described herein is applicable for various purposes. For example, arrays of oligonucleotides can be used for control over the quality of making the array, quantitative or qualitative analysis of nucleic acid molecules, detection of mutations, determination of expression profiles, toxicology tests, and the like.

A. Internal Control in Hybridization or Array Generation In this embodiment, each region of the array contains the same nucleic acid sequence in addition to any other sequence. Thus, one common sequence is located for each element. The consensus sequence can be used as a quality control to generate the array or a control to monitor quality assurance. A “control” sequence can serve as a capture site for a complementary oligonucleotide, which contains or has a detectable label. In this way, the reproducibility of the amount of nucleic acid in each region can be determined. If necessary, the results can be normalized to a control sequence. A control sequence can be incorporated into any of the uses described herein.

B. Probe Quantitation or Typing In this embodiment, 2-100 oligonucleotides per element are immobilized on an array, where each oligonucleotide in the element is a different or related sequence. Be transformed into Preferably, each element has a known or related set of sequences. Hybridization of a labeled probe to such an array allows for characterization of the probe and identification and quantification of sequences contained in the probe population.

A generalized assay format that can be used for the particular application discussed below is
This is a sandwich assay format. In this format, multiple oligonucleotides of a known sequence (eg, 2-100) are immobilized on the same element of the array.
Thus, each element has a known or related set of sequences. Immobilized oligonucleotides are used and nucleic acids (eg, RNA, rRNA, PCR
Product, fragmented DNA) and the signal probes are then hybridized to different portions of the captured target nucleic acid.

Another generalized assay format is a secondary detection system. In this format, an array is used to identify and quantitate the labeled nucleic acids used in the primary binding assay. For example, if the assay produces a labeled nucleic acid, the identity of the nucleic acid can be determined by hybridization to the array. These assay formats are particularly useful when combined with cleavable mass spectrometry tags.

B. Mutation Detection Disease detection is increasingly important in prevention and treatment. Although it is difficult to develop a genetic test for a multifactorial disease, more than 200 known human disorders are caused by a defect in a single gene, often a single amino acid residue change that results in the disease ( Olsen, Biotechnology: An Ind
usry comes of age, National Academic
Press, 1986).

The analysis was based on fertilized egg transplantation (Holding and Monk, Lancet 3:
532, 1989) Cells detached from the respiratory tract or bladder before or in connection with a physical examination (Sidransky et al., Science 252: 706, 1991).
May be implemented. When an unknown gene causes a genetic disease, DNA
Methods for monitoring sequence variants are useful for studying the genetics of a disease through genetic linkage analysis.

[0061] Mutations involving a single nucleotide can be identified in a sample by physical, chemical, or enzymatic means. In general, mutation detection methods fall into two categories: scanning techniques that are appropriate for identifying previously unknown mutations and techniques designed to detect, identify, or quantify known sequence variants. obtain. Several scanning techniques for mutation detection have been developed based on the observation that heteroduplexes of mismatched complementary DNA strands from wild-type and mutant sequences exhibit abnormal migration behavior.

[0062] The methods described herein can be used for mutation screening.
One strategy for detecting mutations in the DNA strand is by hybridization of a test sequence to a target sequence that is a wild-type or mutant sequence. The mismatched sequence has a destabilizing effect on the hybridization of the short oligonucleotide probe to the target sequence (Wetmur. Crit).
. Rev .. Biochem. Mol. Biol. , 26: 227, 1991).
The test nucleic acid source can be genomic DNA, RNA, cDNA or an amplification of any of these nucleic acids. Preferably, amplification of the test sequence is performed first, followed by hybridization with short oligonucleotide probes immobilized on the array. The amplification products can be scanned for many possible sequences by determining the hybridization pattern of the immobilized oligonucleotide probes to the array.

Labels as described herein are generally incorporated into the final amplification product by using labeled nucleotides or by using labeled primers. The amplification product is denatured and hybridized to the array. Unbound products were washed away and the label bound to the array was identified as 1
Detected by two methods. For example, when a cleavable mass spectrometry tag is used.

D. Expression Profiles / Differential Display Mammals, such as humans, have about 100,000 different genes in their genomes, of which a small percentage, perhaps 15%, is any individual It is expressed in cells. The process of normal cell growth and differentiation, and the pathological changes that occur in diseases such as cancer, are all driven by changes in gene expression. Differential display technology allows the identification of genes specific to individual cell types.

Briefly, in differential display, the 3 ′ end portion of the mRNA is amplified and identified based on size. A primer designed to bind to the 5 'boundary of the poly (A) tail for reverse transcription is used, followed by amplification of the cDNA with an upstream arbitrary sequence primer to obtain a subset of mRNA. This differential display method has the ability to visualize all genes (approximately 10,000 to 15,000 mRNA species) expressed in mammalian cells by using a combination of a plurality of primers.

Amplified c to multiple oligonucleotides immobilized on the same element of the array
Hybridization of the DNA allows the identification and quantification of the amplified sequence, and thus the starting mRNA population, where on the array 2-100 oligonucleotides
An element can be immobilized per element, wherein each oligonucleotide in the element is a different or related sequence. Identification of the hybridizing sequence allows for expression profiling of the sequence of the target nucleic acid for which the probe is produced. Expression profiling can be used to measure the regulation of genes and messenger RNA (mRNA) in response to cellular signals, stimuli, and the like. For example, arrays for toxicology testing can be constructed such that individual regions contain sequences complementary to various cytokines (see above). For toxicology testing, the stimulus is generally a small organic compound or small organic molecule that is suspected of being toxic. Or here the toxicology of the small organic molecule should be determined.

As disclosed herein, high-throughput methods for measuring expression of a large number of genes (eg, 1-2000) are disclosed. Within one embodiment of the invention,
(A) using one or more tagged primers to amplify cDNA from a biological sample, wherein the tag correlates with a particular nucleic acid molecule probe and is non-fluorescent A process, which can be detected by spectrometry or potentiometry,
(B) hybridizing the amplified fragments to an array of oligonucleotides as described herein; (c) washing away unhybridized material; and (d) non-fluorescence spectroscopy. Alternatively, a method is provided for analyzing a pattern of gene expression from a selected biological sample, comprising detecting the tag by potentiometry and determining the pattern of gene expression in the biological sample therefrom. Is done.

Tag-based differential display on solid substrates, especially using cleavable mass spectrometry tags, allows the characterization of differentially expressed genes.
This is based on the principle that most mRNA expressed in more than one cell type or sample of interest can be compared directly after amplification of a partial cDNA sequence from a subset of mRNAs. Briefly, oligo dT primers with three single bases anchored are used in combination with a series of optional 13 base oligonucleotides to reverse transcribe and amplify mRNA from cells or samples of interest.
Preferably, at least nine different optional primers are used to monitor the expression of the 15,000 gene. For a complete differential display analysis of two cell populations or two samples of interest, at least 40
A zero amplification reaction is performed. At least 1500 amplification reactions are easily and rapidly performed, with tag-based differential display analysis of two cell types.

E. Single Nucleotide Extension Assay The primer extension method can be used to detect single nucleotides in a nucleic acid template (Sokolov, Nucleic Acids Res., 18: 3661,
1989). In the original format, 30 complementary to the known sequence of the cystic fibrosis gene
Base and 20 base oligonucleotides were extended in the presence of a single labeled nucleotide to correctly identify single nucleotide changes within the gene. This technique is generally applicable to the detection of any single base mutation (Kuppuswamy et al. Proc. Natl. Acad. Sci. USA, 8
8: 1143-1147, 1991).

[0070] Briefly, the method first hybridizes a primer to a sequence adjacent to a known single nucleotide polymorphism. The primed DNA will then be used when the next base in the template is complementary to the labeled nucleotide in the reaction mixture.
DNA polymerase is subjected to conditions that add labeled dNTPs, typically ddNTPs. In a modification, the cDNA is first amplified for a sequence of interest that contains a single base difference between the two alleles. Each amplified product is then annealed with a primer that is 5 ′ one base to the polymorphism, and
By extending by one labeled base (generally a dideoxynucleotide), each allele is analyzed for the presence, absence, or relative amount. The base is incorporated at the 3 'end of the primer only if the correct base is available for the reaction. The extension products are then analyzed by hybridization to an array of oligonucleotides such that the unextended products do not hybridize.

[0071] Briefly, in the present invention, each dideoxynucleotide is labeled with a unique tag. Only one of the four reaction mixtures adds a dideoxy terminator to the primer sequence. If a mutation is present, it is detected by a unique tag on the dideoxynucleotide after hybridization to the array. Multiple mutations can likewise be determined simultaneously by tagging the DNA primer with a unique tag. Thus, the DNA fragment reacts in each of four separate reactants, including a different tagged dideoxy terminator, where the tag correlates with a particular dideoxynucleotide and is determined by non-fluorescence spectroscopy or potentiometry. Can be detected. DNA fragments are hybridized to the array, and unhybridized material is washed away. The tag is cleaved from the hybridized fragment and applied to the respective detection technique (eg, mass spectrometry, infrared spectroscopy, potentiometry or UV
/ Visible spectrophotometry). The detected tags can be correlated with the identity of the particular DNA fragment and variant nucleotide under study.

An array of the invention can be used to detect the products of a single nucleotide extension assay (SNEA). An array (2-100 oligonucleotides per element)
Here, each oligonucleotide in this element is a different or related sequence and is complementary to a given product of a single nucleotide extension assay (SNEA). Preferably, each oligonucleotide sequence used to detect SNEA is included in an adjacent element. For example, SN
The "A" product of EA is contained in element 1, the "T" product of SNEA is contained in element 2, the "C" product of SNEA is contained in element 3, and "G" of SNEA is contained.
The product is included in element 4.

F. Oligonucleotide Ligation Assay Oligonucleotide Ligation Assay (OLA) (Landegen et al., Scie
nce 241: 487, 1988) are used to identify known sequences in very large and complex genomes. OLA principles are based on the ability of ligase to covalently link two diagnostic oligonucleotides when hybridized adjacent to each other with a given DNA target. If the sequence at the junction of the probe is not completely base-paired, the probe will not be bound by the ligase. The ability of the thermostable ligase to discriminate potential one base pair differences when located at the 3 'end of the "upstream" probe offers the opportunity to obtain one base pair resolution (Bar
ony, Proc. Natl. Acad. Sci. USA. 88: 189, 1
991). When tags are used, they are attached to the probe and the probe is ligated to the amplified product. After completion of the OLA, the fragments are hybridized to an array of complementary sequences, the tags are cleaved, and detected by mass spectrometry.

In one embodiment of the method of the invention, a method for determining the identity of a nucleic acid molecule, for example, in a biological sample, or the nucleic acid molecule of choice, utilizing the technology of an oligonucleotide ligation assay Are provided. Briefly, such methods generally involve performing amplification on a target DNA, followed by hybridizing with a 5′-tagged reporter DNA probe and a 5 ′ phosphorylated probe. The sample is incubated with T4 DNA ligase. The DNA strands with the ligated probes are captured on the array by hybridization to the array (where unligated products do not hybridize). The tag is cleaved from the separated fragments and then detected by the respective detection technique (eg, mass spectrometry, infrared spectrophotometry, potentiometry or ultraviolet / visible spectrophotometry).

In the present invention, multiple samples and multiple mutations can be analyzed simultaneously. Briefly, the method comprises amplifying a gene fragment containing the mutation of interest. The amplified product then hybridizes with a consensus probe or two allele-specific oligonucleotide probes (one containing the mutation but not the other), resulting in a 3 ′ of the allele-specific probe. The end is immediately adjacent to the 5 'end of the common probe. This establishes a competitive hybridization-ligation process between the two allele probes and the common probe at each locus.

In one embodiment of the method of the present invention, oligonucleotide ligation assay techniques for simultaneous multiple sample detection are utilized to determine or select the identity of a nucleic acid molecule, for example, in a biological sample. Methods are provided for detecting a nucleic acid molecule that binds. Briefly, such methods generally involve amplifying a target DNA, followed by hybridization with a common probe (untagged) and two allele-specific probes tagged in accordance with the present specification. Is included. This sample is incubated with DNA ligase and the fragments are captured on the array by hybridization. The tag is cleaved from the separated fragments and then detected by the respective detection techniques (eg, mass spectrometry, infrared spectrophotometry, potentiometry, and ultraviolet / visible spectrophotometry).

Landegren et al. (Landegen et al., Science 241: 48)
7, 1988) is a useful technique for the identification of sequences (known) in very large and complex genomes. The principle of the OLA reaction is based on the ability of ligase to covalently link two diagnostic oligonucleotides when they hybridize adjacent to each other at a given DNA target. If the sequence at the probe junction is not a perfect base pair, the probe will not be bound by the ligase. The ability of the thermostable ligase to discriminate potential single base pair differences offers an opportunity for single base pair resolution when located at the 3 'end of the "upstream" probe (Barony, PNAS USA 88: 189, 1991). In tag application, the tag attaches to a probe that is linked to the amplification product. After completion of the OLA, the fragments are hybridized to the array, the tags are cleaved and detected by mass spectrometry.

In another embodiment, the oligonucleotide-ligation assay uses two adjacent oligonucleotides: a “reporter” probe (tagged at the 5 ′ end) and a 5 ′ phosphorylated / 3 ′ tagged. "Anchor" probe. Two oligonucleotides incorporating different tags are annealed to the target DNA, and if completely complementary, the two probes are ligated by T4 DNA ligase. In one embodiment, the 3 'tag is biotin, and the biotinylated anchor probe on immobilized streptavidin is captured and a covalent reporter probe test for the presence or absence of the target sequence is analyzed.

G. Other Assays The methods described herein can also be utilized for genotyping or identifying viruses or microorganisms. For example, F + RNA coliphage may be a useful candidate as an indicator of enteric virus contamination. Genotyping by nucleic acid amplification and hybridization methods is a reliable, fast, simple, and inexpensive alternative to serotyping (Kafatos et al. Nucleic Acids).
s Res. 7: 1541, 1979). Amplification technology and nucleic acid hybridization technology are described in Escherichia coli (Feng, Mol. Cell Probes 7:15).
1, 1993), rotavirus (Sethabutr et al., J. Med Viro).
l 37: 192, 1992), hepatitis C virus (Stuyver et al., J. G.
en Virol. 74: 1093, 1993) and herpes simplex virus (
Matsumoto et al. Virol. Methods 40: 119,19
It has been used successfully to classify various microorganisms, including 92).

Genetic alterations have been described in various experimental mammalian and human neoplasms and show a morphological basis for the sequel of the morphological changes observed in the carcinogenesis event (Vogelstein et al., NEJM 319: 525, 1988). In recent years, with the advent of molecular biology techniques, the lack of arrays on particular chromosomes or mutations in tumor suppressor genes and mutations in several oncogenes (eg, c-myc, c-jun and ras families) have been most studied. It is a thing that is. Previous work (Finkelstein et al., Arch Surg. 128: 526, 1).
993) identified a correlation between a particular type of point mutation in the K-ras oncogene and the stage of diagnosis in colorectal carcinoma. This result suggested that mutation analysis could provide important information about tumor aggressiveness, including the pattern and spread of metastases. The prognostic value of TP53 and K-ras-2 mutation analysis in colonic tertiary carcinoma was shown more recently (Pricolo et al., Am. J. et al.
Surg. 171: 41, 1996). Thus, it is clear that genotyping of tumor and precancerous cells, and detection of specific mutations, will be increasingly important in treating cancer in humans.

The following examples are provided by way of illustration and not limitation.

Examples Example 1: Arrayed chips from commercially available spring probes (arrayy
Preparation of ng tip) This example describes the manufacture and modification of a spring probe tip for use in depositing samples on an array.

The XP54P spring probe is purchased from Osby-Barton (a division of Everett Charles, Pomona, Calif.). The probe was placed "tip-down" on an ultrafine diamond wheel and moved over the stone approximately 0.5 cm with gentle pressure. Under a microscope, remove about 0.005 inch (0.001-0.01 inch) of metal from the end of the tip. The tip end was polished by rubbing the tip across the skin and then washed with water. Chips are stored dry or at -20 ° C
And stored in 50% glycerol. The chip is mounted on the head in an array fashion for use in preparing the array. This head is mounted on a robot arm that has an operation that can be controlled on the Z axis.

Example 2: Preparation of arrays of microspheres on glass slides The deposition of microspheres, which can be easily detected on glass slides, indicates the reproducibility of array formation. In this procedure, 56% glycerol, 0.01 M Tr
is, pH 7.2, 5 mM EDTA, 0.01% sarkosyl and 1%
v / v Fluoresbrite Plain 0.5 μM microspheres (2.5% solid latex) (Polysciences, Warring)
ton, PA) is prepared. Array pins are placed in this solution for 5 seconds.
mm. The microspheres are then repeatedly arranged on a glass slide.
Photomicrographs of the slides are taken under fluorescent light using a filter for fluorescence. FIG.
Indicates that the amount of solution deposited in each region of the array is very consistent. Further, at least 100 deposits can be made per pickup, which are substantially identical.

Example 3 Array Preparation Using a Modified Hydrophilic Spring Probe Sample pick-up, transfer and microdroplet deposition were performed using a liquid transfer device with a hydrophilic surface, especially when the device was used. It is greatly enhanced when it is a modified spring probe. The spring probe becomes hydrophilic, either through the use of a chemical agent that acts to modify the surface of the probe, or by coating the probe with a hydrophilic substance. In a preferred method, the tip of the spring probe is immersed in a 25-200 mM 1,4-dithiothreitol solution, 0.1 M sodium borate for 15 minutes to 2 hours. Dithiothreitol reacts with the gold surface through thiol-gold coordination, which essentially hydroxylates the surface, rendering it hydrophilic.

The arraying solution consists of 56% glycerol and 44% water colored with food blue. The arrayed chip is immersed in the arraying solution for 5 seconds at 5 mm. Next, the chip holding the glycerol is controlled by a robot so that
Print 72 small spots in a 2 × 6 grid. The generated spot is
The diameter is about 100-150 microns and the center-to-center spacing between spots is 200 microns. FIG. 2 shows a CCD camera image of the generated grid. The standard deviation of the spot diameter is about 15%.

Example 4: Colorimetric Detection of Array Oligonucleotides Template Oligonucleotide (75 μl of 0.5 μg / μl)
') In 5 μl of 20 mg in 20 μl of 1 M sodium borate for 30 minutes at room temperature
/ Ml with cyanuric chloride. From this reaction, an arrayed solution is made,
56% glycerol, 56 ng / μl oligonucleotide, 0.06 mM
Consists of sodium borate and 0.3 mg / ml cyanuric chloride. This arrayed chip is immersed for 5 seconds in the arraying solution for 2 seconds. Next, a chip holding this solution is controlled by a robot to print 72 minute spots in a 12 × 6 grid on a silicon wafer coated with polyethyleneimine (PEI). The generated spots are approximately 100-150 microns in diameter and the center-to-center spacing between spots is 200 microns. After arraying, unreacted PEI sites on the wafer are blocked with 100 mg / ml succinic anhydride in 100% n-methylpyrrolidinone for 15 minutes and washed three times with water. The unreacted cyanuric chloride site is maintained at 0 for 15 minutes.
. Block with 0.1 M glycine in 01 M Tris and add Tens buffer (0
. (1 M NaCl, 0.1% SDS, 0.01 M Tris, 5 mM EDTA)
Wash 4 times. The template oligomer was then hybridized with its biotinylated complement (5 ′ biotin-TGTGGATCAGCAAGCAGGAGTATG-3 ′) for 20 minutes at 37 ° C., followed by 6 × Tens and 2 × OHS (0.
06M Tris, 2mM EDTA, 5x Denhardt solution, 6x SSC
(3M NaCl, 0.3M sodium citrate, pH 7.0) 3.68m
Wash with MN-lauroyl sarcosine, 0.005% NP-40). The wafer is then immersed in 0.5 μg / ml of alkaline phosphatase conjugated streptavidin for 15 minutes, then 2 × Tens, 4 × TWS (0.1 M NaCl,
Wash with 0.1% Tween 20, 0.05M Tris). Next, the minute spot is defined as a Vector Blue (Vector Laboratorie).
Chromatography is performed using S. Burlingame, California (according to the protocol of the kit) and imaged with a CCD camera and microscope. FIG. 3 shows the generated image. The obtained micro spots are NIH Image (
National Institute of Health, Bethesd
a, MD) having an intensity value that varies about 15% in diameter and about 10% in diameter.

Example 5 Multiple Oligos in a Single Array Element Two Template Oligos (# 1, 5′-Hexylamine-TGTGGATCAGCA)
AGCAGGAGTATAG-3 ', # 2, 5'-hexylamine-ACTAC
TGATCAGGCGGCCCTTTTTTTTTTTTTTTTTTTTT-3 ′
) At 0.5 μg / μl separately with 5 μl of 20 mg / ml cyanuric chloride and 20 μl of 1 M sodium borate for 30 minutes at room temperature for a total reaction volume of 100 μl
To react. From these two reactions, an arraying solution is made from 56% glycerol and a diluted combination of the two reacted oligos (see table below). Eight arrayed chips were placed in each of the eight arrayed solutions for 5 seconds into 5 mm
Soak. The chip holding this solution is controlled by a robot to print two sets of eight 12 × 6 grids. Each grid is made of polyethyleneimine (PEI)
72 micro spots on a silicon wafer coated with. Each grid represents a single arrayed solution. This generated spot has a diameter of about 100
１５０150 microns and the center-to-center spacing between spots is 200 microns.

Following arraying, unreacted PEI sites on the wafer were blocked with 100 mg / ml succinic anhydride in 100% n-methylpyrrolidone for 15 minutes and washed with water for 3 minutes.
Wash twice. Unreacted cyanuric chloride sites were 0.1 M in 0.01 M Tris.
Block with glycine for 15 minutes and wash with 4 × Tens (0.1 M NaCl, 0.1% SDS, 0.01 M Tris, 5 mM EDTA). Then 2
Twice hybridization is performed. For the first hybridization,
Oligonucleotide 5'-biotin-TGTGGATCAGCAAGCAGGGAGT complementary to oligo # 1 for a set of eight array oligo combinations
Hybridize with ATG-3 '. In the second hybridization, another set of eight array oligos was combined with an oligonucleotide (5'-biotin-AAAAAAAAAAAAAAAAAAAAAG) complementary to oligo # 2.
(GCGCGCCTGATCAGTAGT). These hybridizations are performed by Hybridwell Sealing Covers.
(Research Products International Cor
position, Mount Project, Illinois) for 20 minutes at 37 ° C., followed by 6 × Tens, 2 × OHS (0
. 06M Tris, 2mM EDTA, 5x Denhardt solution, 6x SS
C (3M NaCl, 0.3M sodium citrate, pH 7.0), 3.68
Wash with mM N-lauroyl sarcosine, 0.005% NP-40). After hybridization, the wafer was immersed in 0.5 μg / ml horseradish peroxidase streptavidin for 15 minutes, then 2 × Tens, 4 × T
WS (0.1 M NaCl, 0.1% Tween 20, 0.05 M Tri)
Wash in s). The microspot is then developed using 0.4 mg / ml 4-methoxy 1-naphthol (0.02% hydrogen peroxide, 12% methanol, PBS) and finally washed three times with water.

The set of mixed oligos that hybridized with the complement of oligo # 1 showed the highest color intensity relative to the grid with the highest concentration of oligo # 1, and the lowest with the grid with the lowest concentration of oligo # 1. Shows the color intensity of On the other hand, the set of mixed oligos that hybridized with the complement of oligo # 2 showed the highest color intensity relative to the grid with the highest concentration of oligo # 2 and the lowest with the grid with the lowest concentration of oligo # 2. (See FIG. 4).

[0091]

[Table 2]

[0092] From the foregoing, it is understood that while specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. You. Accordingly, the invention is not limited except as by the appended claims.

[Brief description of the drawings]

FIG. 1 shows micrographs of arrayed microspheres taken under visible light illumination (upper panel) and fluorescent illumination (lower panel).

FIG. 2 shows a CCD camera image of an array generated by a robot using the methodology of the present invention, where the domains have an average diameter of about 100-150 microns and a center-to-center between each spot. The spacing is 200 microns. The standard deviation of this spot diameter is about 15%.

FIG. 3 shows a vector blue (Vector Labo) prepared according to the present invention.
2 shows an array of microspots that were developed using the S. ratoris, Burlingame, California and imaged using a CCD camera and microscope.

FIG. 4 is a schematic diagram showing how two different oligonucleotides, both present in a single array element, can be identified and partially quantified in accordance with the present invention.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU (72) Inventor Moinihan, Kristen USA 9898, Seattle, 9th Ave Avenue Northeast 5026 F-term (reference) 4B024 AA11 AA19 CA01 CA09 CA11 CA12 CA20 HA13 HA14 4B063 QA01 QA13 QQ42 QQ53 QR08 QR32 QR35 QR38 QR42 QR55 QR62 QR84 QS24 QS34 QX02 QX07 QX10

Claims (23)

[Claims] 1. An array of nucleic acid molecules, comprising a solid substrate having a surface comprising distinct regions of nucleic acid molecules, wherein at least one region is selected to have a different sequence. An array containing molecules. 2. The array of claim 1, wherein there are less than 1000 distinct areas. 3. The array according to claim 1, wherein said nucleic acid is an oligonucleotide. 4. The array of claim 1, wherein each region contains at least two nucleic acid molecules having different sequences. 5. The array of claim 1, wherein at least one region contains from 2 to about 100 different nucleic acid sequences. 6. The array of claim 1, wherein the area is between about 20 and about 500 microns in diameter. 7. The array of claim 1, wherein the center-to-center distance between the regions is between about 50 and 1500 microns. 8. The array according to claim 1, wherein said nucleic acid molecule has a known sequence. 9. The method of claim 9, wherein the nucleic acid molecule is covalently attached to a surface of the substrate.
An array according to claim 1. 10. The array of claim 9, wherein said covalent attachment is through an amine linkage. 11. The array of claim 10, wherein the surface of the substrate is coated with poly (ethylene imine). 12. A method of hybridization analysis, comprising: (a) hybridizing a labeled nucleic acid molecule to an array of nucleic acid molecules according to claim 1; and (b) Detecting the label at the region, thereby determining which nucleic acid molecules on the array have hybridized. 13. The method of claim 12, wherein at least one region of said array contains a known sequence and one of said labeled nucleic acid molecules is complementary to said known sequence. 14. The method of claim 13, wherein each region of said array contains said known sequence. 15. The method of claim 12, wherein said labeled nucleic acid molecules comprise nucleic acid molecules having different sequences, each having a different label. 16. The method of claim 12, wherein said label is selected from the group of radioactive molecules, fluorescent molecules, and cleavable mass spectrometry tags. 17. A method for identifying a nucleic acid molecule in a sample, comprising the steps of: (a) hybridizing a labeled oligonucleotide to said nucleic acid molecule to form a duplex; (C) denaturing the duplex; (d) hybridizing the labeled oligonucleotide to the array of oligonucleotides according to claim 2, Wherein the oligonucleotides on the array are complementary to the labeled oligonucleotides; and (e) detecting a label in a region of the array; thereby identifying a nucleic acid molecule in the sample. Including, methods. 18. A method for identifying a nucleic acid molecule in a sample, comprising the steps of:
(A) hybridizing the oligonucleotide to the nucleic acid molecule; (b) extending the oligonucleotide in the presence of a single labeled nucleotide;
(C) denaturing the duplex; (d) hybridizing the labeled oligonucleotide to the oligonucleotide array according to claim 2; Wherein the oligonucleotides on the array are complementary to the labeled oligonucleotides; and (e) detecting a label in a region of the array; thereby identifying a nucleic acid molecule in the sample. how to. 19. The method of claim 18, wherein the oligonucleotides in discrete regions of the array are complementary to extension products of the nucleic acid molecule. 20. A method for identifying a nucleic acid molecule in a sample, comprising the steps of: (a) hybridizing at least two oligonucleotides to the nucleic acid molecule to form a duplex; Wherein at least one oligonucleotide is labeled, and wherein said oligonucleotide hybridizes to an adjacent sequence on said nucleic acid molecule; (b) linking said oligonucleotide; (c) binding said duplex. Denaturing; (d) hybridizing the labeled oligonucleotide to the array of oligonucleotides of claim 2, wherein the oligonucleotides on the array are complementary to the ligated oligonucleotides. And wherein no hybridization occurs to the unligated oligonucleotide, steps; and (e) Labeled detect in the region of b; step of identifying thereby the nucleic acid molecule in said sample; encompasses a method. 21. A method for identifying an mRNA molecule in a sample, comprising the steps of: (a) hybridizing a labeled oligonucleotide to the mRNA molecule to form a duplex; (C) denaturing the duplex; (d) hybridizing the labeled oligonucleotide to the array of oligonucleotides according to claim 2, Wherein the oligonucleotides on the array are complementary to the labeled oligonucleotides; and (e) detecting a label in a region of the array; thereby identifying mRNA molecules in the sample. Including, methods. 22. The method of claim 21, wherein said mRNA molecule is isolated from cells that have been treated with a suspected toxic compound. 23. The method of claim 22, wherein the oligonucleotides on the array are sequences of cytokines.