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Title:  Early leukemia diagnostics using microsphere arrays
United States Patent: 
7,179,598
Issued: 
February 20, 2007

Inventors: 
Nolan; John P. (Santa Fe, NM), Zhou; Feng (Los Alamos, NM)
Assignee: 
The Regents of the University of California (Oakland, CA)
Appl. No.:  10/301,300
Filed: 
November 20, 2002


 

Patheon


Abstract

The present invention provides methods and kits for detecting chromosome translocations. The present invention further provides methods for diagnosing cancer.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and kits for detecting chromosome translocations. The present invention further provides methods for diagnosing cancer, such as, for example, leukemia.

One embodiment of the present invention provides methods for detecting chromosome translocations. A target nucleic acid sequence is amplified from a biological sample. A first oligonucleotide specific for a first region of the translocation is hybridized to the amplified target under conditions in which the first oligonucleotide specifically hybridizes to the first region of the translocation. The first oligonucleotide typically comprises a first capture tag. A second oligonucleotide specific for a second region of the translocation is hybridized to the amplified target under conditions in which the second oligonucleotide specifically hybridizes to the second region of the translocation. The second oligonucleotide typically comprises a second capture tag. The first and second hybridized oligonucleotide sequences are extended to produce a first and a second labeled extended oligonucleotide. In some embodiments, the step of extending comprises adding a labeled dideoxynucleotide to the 3' end of the oligonucleotide. In some embodiments, the dideoxynucleotide has a fluorescent label. The first and second oligonucleotides are hybridized to a first and a second address tag on a solid support under conditions in which the first and second address tags specifically hybridize to the first and second capture tags. The presence of the first and second labeled extended oligonucleotides on the solid support is detected, thereby detecting the presence of the chromosome translocation. In some embodiments, the step of extending occurs on a solid support. In some embodiments, the step of extending precedes the step of hybridizing to an address tag on a solid support. In some embodiments, the first and second capture tags each comprise an oligonucleotide and the first and second address tags each comprise an oligonucleotide. In some embodiments, the first and second hybridized oligonucleotide sequences are extended by a single base. In other embodiments, the first and second hybridized oligonucleotide sequences are extended by multiple bases. In some embodiments, the first and second capture tags each comprise an oligonucleotide and the first and second address tags each comprise an oligonucleotide. In some embodiments, the target nucleic acid is a cDNA.

In some embodiments, a third oligonucleotide specific for a junction region within the translocation is hybridized to the amplified target under conditions in which the third oligonucleotide specifically hybridizes to the junction region. The third oligonucleotide typically comprises a third capture tag. The third hybridized oligonucleotide sequence is extended to produce a third labeled extended oligonucleotide. The third oligonucleotide is hybridized to a third address tag on a solid support under conditions in which the third address tag specifically hybridizes to the third capture tag. The presence of the third labeled extended oligonucleotide on the solid support is detected, thereby detecting the presence of the junction region. In some embodiments, the step of extending occurs on a solid support. In some embodiments, the step of extending precedes the step of hybridizing to an address tag on a solid support. In some embodiments, the third capture tag and the third address tag are oligonucleotides. In some embodiments, the third hybridized oligonucleotide is extended by a single base. In other embodiments, the third hybridized oligonucleotide sequences are extended by multiple bases.

In some embodiments, the solid support is a microparticle. In some embodiments, the microparticle has an optical property, such as, for example, color or fluorescence. In some embodiments, the microparticle is in a microarray. In other embodiments, the microparticle is in a suspension array. In some embodiments, the microparticles are microspheres. In some embodiments, the chromosome translocation is detected is by flow cytometry.

In some embodiments, the biological sample is from a human. In some embodiments, the chromosome translocation is associated with cancer. In some embodiments, the cancer is leukemia.

In some embodiments, the first oligonucleotide and second oligonucleotide comprises a pair of sequences selected from: SEQ ID NOS: 38 and 39, SEQ ID NOS: 40 and 41, SEQ ID NOS: 42 and 43, SEQ ID NOS: 44 and 45, SEQ ID NOS: 45 and 46, SEQ ID NOS: 47 and 48, SEQ ID NOS: 49 and 50, SEQ ID NOS: 51 and 52, and SEQ ID NOS: 53 and 54. In some embodiments, the third oligonucleotide is a sequence selected from: SEQ ID NOS: 93 102.

A further embodiment of the present invention provides a kit for detecting a chromosome translocation. The kit contains a first oligonucleotide specific for a first region of the translocation and a second oligonucleotide specific for a second region of the translocation. In some embodiments, the first and second oligonucleotides comprise the sequences set forth in SEQ ID NOS: 38 54. Each oligonucleotide comprises a capture tag comprising a polynucleotide sequence. In some embodiments, the kit further comprises a third oligonucleotide specific for a junction region within the translocation. In some embodiments, the third oligonucleotide comprises the sequences set forth in SEQ ID NOS:93 102. In some embodiments, the kit further comprises an instruction manual.

Another embodiment of the present invention provides a method of diagnosing cancer. A target nucleic acid is amplified from a biological sample. A first oligonucleotide specific for a first region of the translocation is hybridized to the amplified target under conditions in which the first oligonucleotide specifically hybridizes to the first region of the translocation. The first oligonucleotide typically comprises a first capture tag. A second oligonucleotide specific for a second region of the translocation is hybridized to the amplified target under conditions in which the second oligonucleotide specifically hybridizes to the second region of the translocation. The second oligonucleotide comprises a second capture tag. A third oligonucleotide specific for a junction region within the translocation is hybridized to the amplified target under conditions in which the third oligonucleotide specifically hybridizes to the junction region. The third oligonucleotide comprises a third capture tag. The first, second, and third hybridized oligonucleotide sequences are extended to produce a first, second, and third labeled extended oligonucleotide. The first, second, and third oligonucleotides are hybridized to a first, a second, and a third address tag on a solid support under conditions in which the first, second, and third labeled extended oligonucleotides specifically hybridize to the first, a second, and a third address tag. The presence of the first, second, and third labeled extended oligonucleotides on the solid support is detected, thereby detecting the presence of the chromosome translocation. In some embodiments, the step of extending occurs on a solid support. In some embodiments, the step of extending precedes the step of hybridizing to an address tag on a solid support. In some embodiments, the first, second, and third capture tags each comprise an oligonucleotide, and the second, and a third address tag each comprise an oligonucleotide. In some embodiments, the cancer is leukemia. In some embodiments, the first oligonucleotide and second oligonucleotide comprises a pair of sequences selected from the group consisting of S SEQ ID NOS: 38 and 39, SEQ ID NOS: 40 and 41, SEQ ID NOS:42 and 43, SEQ ID NOS: 44 and 45, SEQ ID NOS: 45 and 46, SEQ ID NOS: 47 and 48, SEQ ID NOS: 49 and 50, SEQ ID NOS: 51 and 52, and SEQ ID NOS: 53 and 54. In some embodiments, third oligonucleotide is a sequence selected from the group consisting of SEQ ID NOS: 93 102. In some embodiments, the solid support is an array. In some embodiments, the solid support is a microarray. In some embodiments, the solid support comprises at least two microparticles. In some embodiments, the microparticles have an optical property. In some embodiments, the microparticles are in a suspension array. In some embodiments, the step of extending comprises adding a labeled dideoxy nucleotide to the 3' end of the oligonucleotide. In some embodiments, the dideoxy nucleotide has a fluorescent label. In some embodiments, the step of detecting is by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides methods and kits for the detection of chromosome translocations. In particular, this invention provide methods for diagnosis of cancers associated with chromosome translocations, such as leukemia and lymphoma.

Identification of Chromosome Translocations

One embodiment of the invention provides methods for detecting chromosome translocations. In particular, the invention provides methods for detecting translocation junctions. In some embodiments, the particular chromosome translocation is identified, then the particular translocation junction associated with that chromosome translocation is identified. In other embodiments of the invention, the chromosome translocation and the particular translocation junction associated with the chromosome translocation are identified simultaneously. In preferred embodiments of the invention, the methods of the present invention are used to diagnose cancers, including, for example, leukemia, lymphoma, breast cancer, renal cancer, peripheral neuroepithelioma, synovial carcinoma, and ganglioneuroblastomas. It will be appreciated by those of skill in the art that the methods of the present invention can be used to diagnose any cancer associated with a chromosome translocation. It will also be appreciated by those of skill in the art that the methods of the present invention can be used in a multiplex strategy to analyze multiple samples in parallel (see, e.g., U.S. Pat. Nos. 6,270,973; 6,280,618; 6,287,766; and 6,361,9506).

A. Amplification of Target Nucleic Acid Sequence

A target nucleic acid sequence (e.g., the region of a chromosome translocation that comprises the translocation junction) is amplified. Amplification of an RNA or DNA template using reactions is well known (see U.S. Pat. Nos. 4,683,195 and 4,683,202; Sambrook and Russell, 2001, supra; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., eds., John Wiley & Sons, Inc. 1994 1997, 2001 version); PCR TECHNOLOGY: PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION (Erlich, ed., 1992); PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Innis et al., eds, 1990)). Methods such as polymerase chain reaction (PCR) and reverse-transcriptase (RT-PCR) can be used to amplify target nucleic acid sequences directly from cDNA, from genomic libraries, cDNA libraries, or from RNA (e.g., total RNA or mRNA).

Genomic DNA can conveniently be isolated from biological samples (e.g., obtained from leukemia or lymphoma patients) and PCR can be used to amplify DNA comprising the chromosome translocation (see, e.g., Sambrook et al., 2001, supra). Briefly, a target nucleic acid comprising the region of a chromosome translocation that comprises the translocation junction, is combined with a primer specific for the first region of the chromosome translocation corresponding to a first gene and a primer specific for a second region of a chromosome translocation corresponding to a second gene, dNTPs, Taq polymerase, and other reaction components. See Innis et al. The first and second gene define the region of the chromosome translocation corresponding to the first chromosome and the second chromosome, respectively. Typically primers are present at a concentration of about 100 nM to about 1 .mu.M, more typically about 150 nM to about 700 nM, more typically about 175 nM to about 500 nM, most typically about 200 nM to about 400 nM. One of skill in the art will appreciate that the primer concentration can be empirically optimized to maximize the efficiency and specificity of the amplification reaction without undue experimentation. For example, some primers will be present at a concentration of about 800 nM. The primers specifically anneal to a first and second region of the chromosome translocation and, if the chromosome translocation is present, amplification of DNA comprising the chromosome translocation occurs.

Alternatively, mRNA can conveniently be isolated from biological samples (e.g., obtained from leukemia or lymphoma patients) and RT-PCR can be used to synthesize cDNA comprising the sequence resulting from the chromosome translocation (see, e.g., Sambrook et al., 2001, supra). It will be appreciated by those of skill in the art that both specific primers and random primers can conveniently be used to synthesize the cDNA sequence resulting from the chromosome translocation. Once the cDNA is synthesized specific primers are used in the extension reactions described below. Briefly, a target nucleic acid comprising the region of a chromosome translocation that comprises the translocation junction, is combined with a primer specific for the first region of the chromosome translocation corresponding to a first gene and a primer specific for a second region of a chromosome translocation corresponding to a second gene, dNTPs, reverse transcriptase, and other reaction components. See Innis et al., supra. The first and second gene define the region of the chromosome translocation corresponding to the first chromosome and the second chromosome, respectively. Typically primers are present at a concentration of about 100 nM to about 1 .mu.M, more typically about 150 nM to about 700 nM, more typically about 175 nM to about 500 nM, most typically about 200 nM to about 400 nM. One of skill in the art will appreciate that the primer concentration can be empirically optimized to maximize the efficiency and specificity of the amplification reaction without undue experimentation. For example, some primers will be present at a concentration of about 800 nM. The primers specifically anneal to a first and second region of the chromosome translocation and, if transcripts of the chromosome translocation are present, amplification of cDNA comprising the chromosome translocation occurs.

In preferred embodiments of the present invention, multiplex amplification is used to amplify the target nucleic acid sequences. One of skill in the art will appreciate that primers that correspond to particular chromosome translocations can be designed for use in the methods of the present invention. For example, multiple sets of the primers shown in FIG. 7 can conveniently be used in a single reaction mixture to amplify any nucleic acid sequences corresponding to E2A-PBX1, MLL-AF4, AML1-ETO, BCR-ABL (p190), BCR-ABL (p210), TEL-AML1, PML-RARA, CBFB-MYH11, SIL-TAL1 chromosome translocations. Multiple sets of the primers shown in FIG. 13 can conveniently be used in a single reaction mixture to hybridize to nucleic acid sequences corresponding to translocation junctions of MLL-AF4 chromosome translocations. It will be appreciated by those of skill in the art that multiplex amplification will amplify only those chromosome translocations or translocation junctions actually present in the target nucleic acid sequences.

The reaction is preferably carried out in a thermal cycler to facilitate incubation times at desired temperatures. Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

B. Detection of Hybridized Oligonucleotide Sequences

Once the cDNA or genomic DNA comprising the chromosome translocation is amplified, the cDNA or genomic DNA can conveniently serve as the template for a primer extension reaction that is used to identify the particular translocation junction (i.e., fusion transcript) present in the sample. Oligonucleotide sequences (e.g., primers) that specifically hybridize to portions of the translocation (e.g., portions of the translocation derived from the first gene, portions of the translocation derived from the second gene, or the translocation junction itself) are annealed to the cDNA and the oligonucleotide sequences are extended.

To increase the specificity of the extension reaction, the oligonucleotide primers that specifically hybridize to portions of the translocation derived from the first gene and portions of the translocation derived from the second gene are designed to specifically hybridize to a region of the first gene and a region of the second gene that are closer to the translocation junction than the primers used to amplify the translocation. In a preferred embodiment, the primers used in the extension reaction bind to a region of the translocation that does not overlap with the region of the translocation bound by the primers used to amplify the translocation. For example, if 20 base primer is used for the amplification reaction, the primer for the extension reaction will bind to a portion of the translocation that does not overlap with the region of the translocation bound by the 20 base primer. It will be appreciated by those of skill in the art, however, that some overlap between the extension primers and the amplification primers will not substantially affect the specificity of the extension reaction.

Typically, oligonucleotide primers that specifically hybridize to the translocation junction itself are used to identify translocation junctions. The primers are designed such that that they will only hybridize to the amplified cDNA or genomic DNA if the translocation junction is present; if the translocation junction is not present in the amplified cDNA or genomic DNA, the primers will not hybridize to it. Typically, the primers are designed so that the middle of the primer hybridizes to the translocation junction. For example, a 20 nucleotide primer may be aligned to hybridize to a translocation junction so that 10 nucleotides hybridize to the portion of the translocation derived from first gene and 10 nucleotides hybridize to the portion of the translocation derived from second gene. One of skill in the art will appreciate, however, that such a precise alignment is not a critical aspect of the invention. For example, 20 nucleotide primer will hybridize to a translocation junction so that 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides hybridize to the portion of the translocation derived from first gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides hybridize to the portion of the translocation derived from second gene, respectively. Primers can be designed using any means known in the art, including, for example, commercially available and custom software such as OLIGO6. Typically, the software will use algorithms so that annealing temperatures are close to melting temperature. Preferably, all of the primers are designed so that they are compatible in a multiplex extension reaction.

The oligonucleotide sequences (e.g., primers) comprise a sequence that hybridizes to a chromosome translocation and a capture tag. In some embodiments, a capture tag is a sequence that does not hybridize to a chromosome translocation. A capture tag may also be the primer itself. Typically, a capture tag is located at the 5' end of the oligonucleotide. A capture tag can be any length. Typically, a capture tag is about 10 to about 40 nucleotides in length, more typically about 15 to about 30 nucleotides in length, even more typically at least 18 to about 21 nucleotides in length. In some embodiments of the present invention, the capture tag and the oligonucleotide are separated by a linker. Suitable linkers include, for example, carbon linkers as described in, e.g., Lukhtanov et al., Bioconjug. Chem., 7: 564 567 (1996). One of skill in the art will appreciate that the linker can be any length and will be designed to minimize interference with the interaction between the capture tag and the address tag. Typically, the carbon linker will be between about 2 and about 25 carbons in length, more typically between about 4 and about 20 carbons in length, even more typically between about 5 and about 18 carbons in length, most typically between about 7 and about 15 carbons in length. Typically, capture tags specifically hybridize to address tags, a moiety, typically an oligonucleotide sequence that, specifically hybridizes to the capture tag. An address tag can be any length. Typically, an address tag is at least 10 nucleotides in length, more typically at least 15 nucleotides in length, even more typically at least 20 nucleotides in length. In some embodiments of the present invention, the-address tag comprises a nucleotide sequence that is the reverse complement of the primer used in an extension reaction to identify a particular translocation junction, as described above.

A labeled, extended oligonucleotide is formed from each oligonucleotide only if the respective translocation or translocation junction was present in the original nucleic acid sample. The oligonucleotides corresponding to different translocation junctions further comprise capture tags. Each capture tag comprises a unique sequence that is complementary to all or part of a corresponding address oligonucleotide. The use of a unique capture tag for each translocation junction increases the efficiency of detection of the chromosome translocation. For example universal arrays can be used with any primers or combination or primers provided that the primers comprise a capture tag that binds an address tag on the array. Furthermore, the use of capture tags eliminates interference from unreacted amplification primers and partially extended products. 1. Extension of the Hybridized Oligonucleotide Sequences

In some embodiments of the present invention, the hybridized oligonucleotide sequences are extended by a single base using single base extension (SBE) (see, e.g., Sylvanen et al, Genomics 8:684 692 (1990); U.S. Pat. Nos. 5,846,710 and 5,888,819; Pastinen et al., Genomics Res. 7(6):606 614 (1997)). SBE is a technique that utilizes an extension primer that hybridizes to the target nucleic acid. A polymerase (generally a DNA polymerase) is used to extend the 3' end of the primer with a nucleotide analog labeled a detection label as described herein. Based on the fidelity of the enzyme, a nucleotide is only incorporated into the extension primer if it is complementary to the adjacent base in the target nucleic acid. Often, the nucleotide is derivatized such that no further extensions can occur after a single nucleotide is added.

The reaction is initiated by combining the target sequence (e.g., the amplified cDNA or genomic DNA comprising the chromosome translocation), primers specific for the translocation junctions, and a solution comprising a dideoxynucleoside-triphosphate (ddNTP), e.g., ddATP, ddCTP, ddGTP and ddTTP or ddUTP. Typically, the ddNTP is labeled, e.g., with a molecule or moiety that is capable of being detected. The labels may be the same or different. Suitable labels include, for example, radioisotopes (e.g., .sup.32P, .sup.35S, .sup.3H, .sup.125I), fluorophores, chemiluminophores, colloidal particles, fluorescent dyes (see, e.g., U.S. Pat. No. 6,403,807) and non-fluorescent dyes.

In addition to a first nucleotide, the extension solution also comprises an extension enzyme, generally a DNA polymerase. Suitable DNA polymerases include any conventional or thermostable DNA polymerase such as, for example, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), Thermosequenase (Amersham), T5 DNA polymerase and Phi29 DNA polymerase. If the NTP is complementary to the base of the translocation junction, which is immediately adjacent (i.e., one nucleotide away from the junction) to the oligonucleotide annealed to the target nucleic acid, the extension enzyme will add the NTP to the oligonucleotide. Thus, the oligonucleotide is modified, i.e. extended, to form a modified oligonucleotide which can be detected as described below.

In some embodiments of the present invention, the first and second hybridized oligonucleotide sequences are extended by multiple bases using "labeled chain extension." The reaction is initiated by combining the target sequence (e.g., the amplified cDNA or genomic DNA comprising the chromosome translocation), primers specific for the translocation junction and comprising a capture tag, and a solution comprising a labeled (e.g., with a fluorescent label) deoxynucleoside-triphosphate (dNTPs), e.g., dATP, dCTP, dGTP and dTTP or dUTP. Multiple labeled dNTP's will be added to the end of the oligonucleotide, thus increasing the amount of signal present on the extended oligonucleotide sequence. 2. Ligation

In some embodiments of the present invention, ligation is used to detect the presence of translocation junctions. The first and second hybridized oligonucleotide sequences are typically designed to hybridize to two adjacent portions of a chromosome translocation. For example, the first oligonucleotide may specifically hybridize to the translocation junction and the second oligonucleotide may specifically hybridize to a conserved region of one gene of the chromosome translocation adjacent to the translocation junction. Alternatively, the two oligonucleotides may specifically hybridize to adjacent portions of the translocation junction. If the translocation junction is present, both oligonucleotides will hybridize to the target nucleic acid sequence and can be ligated to each other (see, e.g., U.S. Pat. No. 6,287,766). Typically one of the oligonucleotides is labeled, e.g., with a fluorescent label; and the other oligonucleotide comprises a capture tag. The ligated primers can be hybridized to an address tag and the chromosome translocation detected as described below. 3. Multiplex Reactions

In preferred embodiments of the present invention, multiplex extension is used to extend the hybridized oligonucleotide sequences (see, e.g., U.S. Pat. Nos. 6,287,766; 5,814,491). One of skill in the art will appreciate that primers that correspond to particular chromosome translocations and translocation junctions can be designed for use in the methods of the present invention. For example, multiple sets of the oligonucleotides shown in FIG. 9 can conveniently be used in a single reaction mixture to extend oligonucleotide sequences corresponding to E2A-PBX1, MLL-AF4, AML1-ETO, BCR-ABL (p190), BCR-ABL (p210), TEL-AML1, PML-RARA, CBFB-MYH11, SIL-TAL1 chromosome translocations and multiple sets of the primers shown in FIG. 14 can conveniently be used in a single reaction mixture to amplify nucleic acid sequences corresponding to MLL-AF4 chromosome translocations and translocation junctions.

As will be appreciated by those in the art, the configuration of both of the extension reactions can take on several forms. For example, the extension reactions may be done in solution, the labeled, extended oligonucleotides can be bound via their capture tags to address tags bound to microparticles, and the labeled, extended oligonucleotides can be detected. Alternatively, the extension reaction can occur on a solid support. For example, the oligonucleotides can be bound via their capture tags to address tags bound to microparticles. The oligonucleotides bound to microparticles can then be added to an extension reaction mixture comprising the amplified target nucleic acid. After the extension reaction, any labeled, extended oligonucleotides attached to the microparticles can be detected.

IV. Detecting the Chromosome Translocation

Once the oligonucleotides annealed to the target nucleic acid have been extended or the oligonucleotides have been ligated, the labeled extended oligonucleotides or ligated oligonucleotides are detected, thereby detecting the presence of the chromosome translocation and identifying particular translocation junctions. The labeled, extended oligonucleotides or ligated oligonucleotides can be detected using any means known in the art. Typically, the labeled extended oligonucleotides or ligated oligonucleotides are bound to a solid support, e.g., a microparticle and detected. Typically, microparticles useful in the methods of the present invention possess an extrinsic optical property such as, for example, color, fluorescence, luminescence, or brightness. Exemplary microparticles are described in, e.g., WO 02/065123; WO 02/064829; WO 01/25002; WO 01/25758; U.S. Patent Publication No. 20020119470 A1.

Many methods for immobilizing nucleic acids (e.g., address oligonucleotides) onto a variety of solid surfaces or solid supports are known in the art. In preparing the surface, a plurality of different materials may be employed, particularly as laminates, to obtain various properties. For example, proteins (e.g., bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt's solution) can be employed to avoid non-specific binding, simplify covalent conjugation, enhance signal detection or the like. If covalent bonding between a compound and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like.

Often, the microparticles are encoded or positioned to form a microarray. Among the various microarray-based molecular analysis tools, the use of microparticle arrays is gaining attention, especially for high throughput applications. The concept of using microspheres and flow cytometry to perform multiplexed assays was initially proposed by Horan and Wheeless, Science 198(4313):149 57 (1977), using different sized microspheres, and further developed recently using different colored microspheres (Fulton et al., Clinical Chemistry 43:1749 1756 (1997)). Microparticles may have different optical properties (e.g., fluorescence or light scatter) that can be discriminated by the flow cytometer. For example, using two fluorescent dyes incorporated into microparticles in different amounts, nearly a hundred different optically encoded microparticles can be identified. A specific molecular reaction is configured on the surface of each microparticle subset. After the reaction, microparticles are identified and the fluorescent signals for each reaction are measured using flow cytometry. Microsphere arrays have been successfully used for immunoassays, single nucleotide polymorphism (SNP), genotyping, bacterial signature detection, and detection of DNA or RNA viruses (Fulton et al., 1997, supra; Cai et al, Genomics 66:135 143 (2000); Nolan et al. 47th Annual Meeting of the American Society of Human Genetics, Oct. 28 Nov. 1, 1997 Baltimore Md.; Iannone et al., Cytometry 39:131 140 (2000); Vignali, J. Immunological Methods 243:243 255 (2000); Armstrong et al., Cytometry 40:102 108 (2000); and Defoort et al., J. Clinical Microbiology 38:1066 1071(2000)).

In a preferred embodiment, the microparticles are in a suspension array. "Suspension array" as used herein refers to an array comprising microparticles suspended in fluid (see, e.g., Nolan and Skar, Trends in Biotech. 20(1):9 12 (2002)). Typically, each microparticle in the array has a distinct optical property. Typically, a suspension array to be hybridized to the labeled, extended oligonucleotide sequences has about 10.sup.4 to about 10.sup.10 microparticles per ml, more typically about 10.sup.6 to about 10.sup.8 microparticles per ml, most typically about 10.sup.7 microparticles per ml. Any aqueous fluid that will not interfere with the optical analysis of the microparticles can be used in the array. Suitable fluids for a suspension array include, for example, saline, phosphate buffered saline, Tris buffer, or culture media for mammalian cells.

Analysis of the microparticles in a suspension array is typically done by flow cytometry (see, e.g., U.S. Pat. No. 6,287,766). Typically, the microparticles in the suspension array are diluted at least 10 fold, more typically at least 50 fold, most typically at least 100 fold before flow cytometry. Any aqueous fluid that will not interfere with the optical analysis of the microparticles can be used to dilute the microparticles before analysis. Suitable fluids for flow cytometry analysis include, for example, saline, phosphate buffered saline, Tris buffer, or culture media for mammalian cells. One of skill in the art will be able to select the degree of dilution and suitable fluids without undue experimentation.

One of skill in the art will appreciate that other methods of analyzing microparticles include, for example, fiber optic arrays in which beads are loaded into the end of a optic fiber and read using image analysis as is known in the art (see, e.g., U.S. Pat. Nos. 6,429,027; 6,396,995; 6,355,431; 5,250,264; and 5,244,636 and dipstick arrays in which beads are packed into a monolayer and read using image analysis as is known in the art (see, e.g., U.S. Pat. Nos. 6,197,598; 6,168,956; 6,146,833; 6,110,749; 6,087,184; 6,069,014; 6,017,767; 6,008,059; 5,998,220; and 5,981,185).

In some embodiments of the invention, the solid supports are include, for example, planar solid support (e.g. a glass surface, a membrane, etc.) or a high density microarray. Preparation and use of high density spotted arrays is described in, e.g., U.S. Pat. Nos. 6,428,957; 5,807,522; 5,143,854; Fodor et al., Science 767 773 (1991); WO 90/15070 and WO 92/10092.

In one embodiment of the invention, the labeled extended oligonucleotides are detected using flow cytometry. Methods and apparati for flow cytometry are described in, e.g., U.S. Pat. Nos. 6,382,228; 6,357,307; 6,287,766; 6,256,096; 6,248,590; and 5,540,494. For example, fluorescent signals on microparticles can be measured using a fluorescence activated cell sorter in conjunction with appropriate acquisition and analysis software. Colored microparticles can be gated using color compensation as is known in the art. The median fluorescence channel can be recorded for each subset of microparticles.

V. Kits

The present invention further provides kits for use in detecting chromosome translocations and diagnosing cancer. Such kits typically comprise a first oligonucleotide specific for a first region of the translocation and a second oligonucleotide specific for a second region of the translocation. In some embodiments, each oligonucleotide comprises a capture tag comprising a polynucleotide sequence. In some embodiments, the first and second oligonucleotides comprise the sequences set forth in SEQ ID NOS: 38 54. In some embodiments, the kits also include a third oligonucleotide specific for a junction region within the translocation. In some embodiments, the third oligonucleotide comprises the sequences set forth in SEQ ID NOS:93 102. Additional components of the kits may be compounds, reagents, containers, equipment, and/or instructions for using the components in accordance with the methods disclosed herein. For example, one container within a kit may contain address tags or address tags bound to a solid support such as, a microparticle. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Other additional components that may be present within such kits include a reagent or container to facilitate the detection of a labeled, extended oligonucleotide using the methods of the present invention.
 


Claim 1 of 6 Claims

1. A method of detecting a chromosome translocation associated with leukemia, the method comprising: (a) amplifying a target nucleic acid sequence from a biological sample; (b) hybridizing a first oligonucleotide comprising the sequence set forth in SEQ ID NO: 40 to the amplified target under conditions in which the first oligonucleotide specifically hybridizes to a first region of the translocation, wherein the first oligonucleotide comprises a first capture tag; (c) hybridizing a second oligonucleotide comprising the sequence set forth in SEQ ID NO: 41 to the amplified target under conditions in which the second oligonucleotide specifically hybridizes to a second region of the translocation, wherein the second oligonucleotide comprises a second capture tag; (d) extending the first and second hybridized oligonucleotide sequences to produce a first and a second labeled extended oligonucleotide; (e) hybridizing the first and second oligonucleotides to a first and a second address tag on a solid support under conditions in which the first and second address tags specifically hybridize to the first and second capture tags; and (f) detecting the presence of the first and second labeled extended oligonucleotides on the solid support, thereby detecting the presence of the chromosome translocation associated with leukemia.

 

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