| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 31.12.02 |
| PATENT TITLE |
Methods of using semiconductor nanocrystals in bead-based nucleic acid assays |
| PATENT ABSTRACT | Methods, compositions and articles of manufacture for assaying a sample for a target polynucleotide and/or an amplification product therefrom are provided. The methods comprise contacting a sample suspected of containing the target polynucleotide with a polynucleotide that can bind specifically thereto; this polynucleotide is conjugated to a substrate, preferably an encoded bead conjugate. An amplification reaction can first be used to produce the amplification product from the target polynucleotide so that it can be used to indirectly assay for the target polynucleotide. An amplification product detection complex and method of forming the same are also provided. The methods are particularly useful in multiplex settings where a plurality of targets are present. Amplification product assay complexes and amplification product assay arrays are also provided, along with methods of forming the same. Kits comprising reagents for performing such methods are also provided |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | March 22, 2001 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An encoded bead conjugate comprising: a microsphere comprising a spectral code comprising a first semiconductor nanocrystal having first fluorescence characteristics; and a first polynucleotide having a proximal end and at least one distal end wherein the first polynucleotide is linked to the microsphere at the proximal end, wherein the first polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions, and wherein the first polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop, and wherein at least part of the third region is complementary to at least a part of a first target polynucleotide, and wherein the first polynucleotide can preferentially hybridize to the first target polynucleotide and thereby disrupt formation of the stem-loop structure under at least one set of hybridization conditions. 2. The encoded bead conjugate of claim 1, wherein the spectral code further comprises a second semiconductor nanocrystal having second fluorescence characteristics. 3. The encoded bead conjugate of claim 1, wherein the first semiconductor nanocrystal comprises a core selected from the group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS, Ge, Si, Pb, PbSe, and a mixture thereof. 4. The encoded bead conjugate of claim 3, wherein the core is CdSe. 5. The encoded bead conjugate of claim 1, wherein the semiconductor nanocrystal comprises an outer shell. 6. The encoded bead conjugate of claim 1, wherein the proximal end of the polynucleotide is the 5' end of the polynucleotide. 7. The encoded bead conjugate of claim 1, wherein the proximal end of the polynucleotide is the 3' end of the polynucleotide. 8. The encoded bead conjugate of claim 1, wherein an internal position of the polynucleotide is the proximal end, and the polynucleotide has a plurality of distal ends. 9. The encoded bead conjugate of claim 1 wherein the bead conjugate further comprises: (i) a first quencher, (ii) a first fluorophore, wherein the first quencher and first fluorophore are located in the conjugate such that the first quencher quenches a fluorescence emission from the first fluorophore either under a first hybridization state when the first polynucleotide is not hybridized to the first target polynucleotide or under a second hybridization state when the first polynucleotide is hybridized to the first target polynucleotide, but not under both hybridization states; and (iii) a second polynucleotide having a proximal end and at least one distal end wherein the second polynucleotide is linked at its proximal end to the microsphere, and wherein the second polynucleotide is linked to a second fluorophore, wherein the second polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions, wherein at least a part of the third region of the second polynucleotide is complementary to at least a part of a second target polynucleotide, wherein the second polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop in the absence of hybridization to the second target polynucleotide, wherein the second polynucleotide can preferentially hybridize to the second target polynucleotide and the stem-loop structure is not formed under at least one set of hybridization conditions, and wherein the fluorescence emission from the second fluorophore is quenched either under a third hybridization state when the second polynucleotide is not hybridized to the second target polynucleotide or under a fourth hybridization state when the second polynucleotide is hybridized to the second target polynucleotide, but not under both third and fourth hybridization states. 10. The encoded bead conjugate of claim 9, wherein the first fluorophore is a second semiconductor nanocrystal having second fluorescence characteristics. 11. The encoded bead conjugate of claim 9, wherein the first fluorophore is a dye. 12. The encoded bead conjugate of claim 11, wherein the first fluorophore is also the quencher and self-quenches when the first polynucleotide is not hybridized to the first target polynucleotide. 13. The encoded bead conjugate of claim 9, wherein the first quencher is linked to the microsphere and the first fluorophore is linked to the first polynucleotide at or nearer the distal end. 14. The encoded bead conjugate of claim 9, wherein the first quencher is linked to the first polynucleotide at or nearer the proximal end. 15. The encoded bead conjugate of claim 9, wherein the first quencher is selected from DABCYL, BHQ-1, BHQ-2, BHQ-3, a metal nanoparticle, and a second semiconductor nanocrystal. 16. The encoded bead conjugate of claim 9, wherein the first quencher quenches the fluorescence emission from the first fluorophore under the first hybridization state. 17. The encoded bead conjugate of claim 9, wherein the first quencher quenches the fluorescence emission from the first fluorophore under the second hybridization state. 18. The encoded bead conjugate of claim 9, wherein the second fluorophore is a dye that self-quenches and is linked to the second polynucleotide so that the dye is quenched in one, but not both, of the third and fourth hybridization states. 19. The encoded bead conjugate of claim 9, wherein the second fluorophore is linked to the second polynucleotide at or nearer its distal end. 20. The encoded bead conjugate of claim 19, wherein the first quencher is linked to the microsphere and can quench both the first and second fluorophores when the first and second polynucleotides are not hybridized to their respective target polynucleotides. 21. The encoded bead conjugate of claim 19, wherein a second quencher is linked to the second polynucleotide at or nearer its proximal end. 22. The encoded bead conjugate of claim 9, wherein the second fluorophore is linked to the second polynucleotide at or nearer its proximal end. 23. The encoded bead conjugate of claim 22, wherein a second quencher is linked to the second polynucleotide at or nearer its distal end. 24. A method of assaying for a first target polynucleotide in a sample, comprising: contacting the sample suspected of containing the first target polynucleotide with the encoded bead conjugate of claim 10 under a first set of hybridization conditions in which the first polynucleotide can hybridize to the first target polynucleotide; wherein a change in fluorescence characteristics of the conjugate results upon hybridization of the first target polynucleotide to the first polynucleotide; identifying the encoded bead conjugate by its spectral code; and determining if a change in fluorescence characteristics of the conjugate has resulted from said hybridization. 25. The method of claim 24, wherein identifying the encoded bead conjugate by its spectral code occurs prior to determining if a change in fluorescence characteristics has resulted. 26. The method of claim 24, wherein identifying the encoded bead conjugate by its spectral code occurs subsequent to determining if a change in fluorescence characteristics has resulted. 27. The method of claim 24, wherein identifying the encoded bead conjugate by its spectral code occurs simultaneously with determining if a change in fluorescence characteristics has resulted. 28. The method of claim 24, wherein the sample is assayed for the presence of the target polynucleotide. 29. The method of claim 24, wherein the sample is assayed for the amount of the target polynucleotide. 30. The method of claim 24, wherein the change in fluorescence characteristics comprises the addition of a fluorophore to the conjugate. 31. The method of claim 24, wherein the change in fluorescence characteristics comprises the removal of a fluorophore from the conjugate. 32. The method of claim 24, wherein the change in fluorescence characteristics comprises the quenching of a fluorophore. 33. The method of claim 24, wherein the change in fluorescence characteristics comprises the removal of quenching from a fluorophore. 34. The method of claim 24, wherein the target polynucleotide is labeled with a fluorophore which upon hybridization of the target polynucleotide to the first polynucleotide changes the fluorescence characteristics of the encoded bead conjugate by adding a fluorescence emission. 35. A kit comprising: the encoded bead conjugate of claim 1; a housing for retaining the encoded bead conjugate; and instructions provided with said housing that describe how to use the components of the kit to assay a sample for a target polynucleotide. 36. A kit comprising: the encoded bead conjugate of claim 9; a housing for retaining the encoded bead conjugate; and instructions provided with said housing that describe how to use the components of the kit to assay a sample for a target polynucleotide. 37. The encoded bead conjugate of claim 3, wherein the mixture is an alloy thereof. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
TECHNICAL FIELD This invention relates to methods, articles and compositions for the analysis of polynucleotides in a sample. BACKGROUND OF THE INVENTION Michael Adams-Conroy died at the age of nine of the highest overdose of Prozac.RTM. on record, seven times higher than any previously known. His parents were investigated for homicide and his two siblings were removed from their custody by social welfare workers. Autopsy results, however, showed no pills in his stomach even though he would normally have had to ingest a huge number in order to reach the levels of drug found in his blood. Acute lymphocytic leukemia (ALL) affects thousands of children each year in the United States. Treatment with chemotherapeutic agents now leads to remission in over 90% of the cases. 6-mercaptopurine (6-MP) is one agent used to treat ALL. However, the normal treatment dose of 6-MP is toxic for one in 300 patients and can kill rather than cure. Adverse reactions to therapeutic drugs have been estimated to kill over 100,000 hospitalized patients in the U.S. each year (Lazarou et al., JAMA Apr. 15, 1998;279(15):1200-1205). This figure does not include intentional overdoses leading to hospitalization which ultimately prove fatal. An additional 2.2 million serious nonfatal adverse drug reactions have been estimated to occur. The problem of the varied responses of individual patients to particular drug therapies is well known, but little progress has been made towards anticipating patients' varied drug metabolisms prior to treatment. The standard approach in administering drugs has been to prescribe the recommended dosage for a given condition to an affected patient, in some cases adjusting for the patient's weight. If the patient does not improve, the dosage is increased or an alternative drug is tried. Conversely, if adverse side effects occur, the dosage may be lowered or an alternative drug employed. Drugs which exhibit serious side effects may never be approved by regulatory authorities or, if approved before such side effects are identified, can be withdrawn from the market if even a small percentage of treated patients are so affected. This can occur despite the fact that such drugs may have great therapeutic benefit in the majority of patients. The 6-MP sensitivity exhibited by rare ALL patients has been linked to a deficiency in thiopurine S-methyltransferase (TPMT) activity (Krynetski et al., Pharm Res 1999 16(3):342-349). Patients deficient in this enzyme can be treated with lower doses of 6-MP to achieve the same therapeutic plasma levels while avoiding adverse toxicity if the prescribing physician is aware of the metabolic deficiency. Metabolism of similar drugs such as azathioprine and thioguanine used in the treatment of rheumatoid arthritis, leukemia and Crohn's disease is also affected in patients who are deficient in TPMT. Cytochrome p-450 CYP2D6 (debrisoquin hydroxylase) is the primary enzyme responsible for human metabolism of fluoxetine (Prozac.RTM.), as well as codeine, amphetamines, methadone, and several antidepressants and neuroleptics. At least twenty variants of the CYP2D6 gene are now known to result in poor metabolism of Prozac.RTM. and other drugs (Wong et al., Ann Acad Med Singapore 2000 29(3):401-406). Approximately 7-10% of Caucasians are poor metabolizers of Prozac.RTM., and reach higher than expected plasma levels when treated with a standard dosage. Michael Adams-Conroy was one such patient, but he was never tested to determine whether he harbored any of the CYP2D6 variants resulting in slow metabolism of Prozac. Instead, because of his diminished response to Prozac.RTM., as typically occurs with chronic use, his dosage was gradually increased to maintain control over his symptoms. Side effects associated with Prozac.RTM. toxicity such as nausea and dizziness were instead attributed to migraines. Only after Michael's death were his tissues tested and shown to contain CYP2D6 variants which contributed to a toxic accumulation of Prozac.RTM. and its metabolites in his blood (Sallee et al., J. Child Adolesc. Psychopharmacol. 2000 Spring; 10(1):27-34). Potentially fatal adverse drug reactions are now known to be associated with altered metabolism by patients harboring variants in a number of genes, including in the NAT2 gene affecting isoniazid metabolism, in the CYP2C9 gene affecting warfarin metabolism, in the DPD gene affecting 5-fluorouracil metabolism, and in the KCNE2 gene affecting clarithromycin metabolism (Grant et al., Pharmacology 2000 61(3):204-211; Taube et al., Blood 2000 96(5):1816-1819; Meinsma et al., DNA Cell Bio 1995 14(1):1-6; Sesti et al., Proc Natl Acad Sci USA 2000 97(19):10613-10618). There is a need in the art for methods of analyzing samples for particular polynucleotides, and for devices, compositions and articles of manufacture useful in such methods. SUMMARY OF THE INVENTION Methods, compositions and articles for assaying a sample for a target polynucleotide or an amplification product therefrom are provided. The methods involve contacting a sample suspected of containing a target polynucleotide with an encoded bead conjugate comprising a probe polynucleotide and a spectral code comprising a semiconductor nanocrystal. The probe polynucleotide can be in a form suitable for performing a cleavase assay, or can be a molecular beacon, or can have an unlabeled stem-loop structure. Binding of the probe polynucleotide to the target polynucleotide results in a change in fluorescence characteristics of the encoded bead conjugate. Amplification reactions can be incorporated into the methods. In one variation of the method, an unlabeled probe polynucleotide that can form a stem-loop structure is employed which can be conjugated to any form of substrate and used to assay for a labeled amplification product. Binding of the probe polynucleotide to the labeled amplification product unfolds the stem-loop structure and results in the production of an amplification product assay complex. Where a plurality of different unlabeled probe polynucleotides are attached to the substrate, binding of a plurality of corresponding different labeled amplification products results in the formation of an amplification product assay array. Kits comprising reagents useful for performing the methods of the invention are also provided. The methods are particularly useful in multiplex settings where a plurality of different conjugates are used to assay for a plurality of different target polynucleotides. The large number of distinguishable semiconductor nanocrystal labels allows for the simultaneous analysis of multiple labeled target polynucleotides, along with multiple different encoded bead conjugates. Methods of the invention can optionally be implemented in a homogeneous format. This allows for higher assay throughput due to fewer manipulations of the sample, and decreased cross-contamination resulting in more reliable assays and less downtime from cross-contamination. If real time monitoring is used, the entire assay can be disposed of without opening a sealed assay chamber such as a sealed microplate, thus further decreasing the risk of cross-contamination |
| PATENT PHOTOCOPY | Available on request |
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