Claims
- 1. A method for preparing a labeled oligonucleotide as a member of a family of labeled oligonucleotides each having a different mobility, said method comprising:
synthesizing an oligonucleotide using an automated synthesizer employing a solid surface; at the terminus of said synthesized oligonucleotide while bound to said surface sequentially adding at least two of a mass-modifying region, a charge-modifying region and a detectable region, using the automated synthesizer where two of said regions can be combined in a single region; to produce one member of a family of labeled oligonucleotides.
- 2. A method according to claim 1, wherein said oligonucleotides and regions are joined by phosphate linkages.
- 3. A method according to claim 2, wherein said synthesizing and adding employs phosphoramidites for producing said member.
- 4. A method for preparing a labeled oligonucleotide as a member of a family of labeled oligonucleotides each having a different mobility, said method comprising:
synthesizing an oligonucleotide using an automated synthesizer employing a solid surface; at the terminus of said synthesized oligonucleotide while bound to said surface sequentially adding a mass-modifying region, a charge-modifying region and a detectable region, using the automated synthesizer where two of said regions can be combined in a single region; wherein said mass-modifying group is a neutral molecule, said charge-modifying region comprises at least one amino acid, at least one carboxyl substituted polyester, or at least one phosphate ester, and said detectable region is a fluorescer. to produce one member of a family of labeled oligonucleotides
- 5. A method according to claim 4, wherein said mass-modifying group is a hydocarbon, a halohydrocarbon, a substituted aromatic compound, or comprises at least one alkyleneoxy.
- 6. A method according to claim 4, wherein said charge-modifying group comprises amino acids.
- 7. A method according to claim 4, wherein said charge-modifying region comprises a phosphate group and said regions are joined by phosphate groups, wherein said phosphate groups comprise said charge-modifying region.
- 8. A method according to claim 4, wherein said method is repeated at least 5 times to produce 5 different labeled oligonucleotides.
- 9. A method for preparing a family of labeled oligonucleotides as members of a family of labeled oligonucleotides each having a different mobility, said method comprising:
synthesizing an oligonucleotide using an automated synthesizer employing a solid surface and phosphoramidite chemistry; at the terminus of said synthesized oligonucleotide while bound to said surface sequentially adding a mass-modifying region, a charge-modifying region and a detectable region, using the automated synthesizer and phosphoramidite chemistry, where two of said regions can be combined in a single region; wherein said mass-modifying region is a neutral group, said charge-modifying region comprises at least one amino acid, at least one carboxyl substituted polyester, or at least one phosphate ester, and said detectable region is a fluorescer; to produce one member of a family of labeled oligonucleotides, and repeating said synthesizing and adding to produce additional members of said family.
- 10. A method according to claim 9, wherein said charge-modifying region comprises a phosphate.
- 11. A method according to claim 9, wherein said neutral group is an alkylene.
- 12. A method according to claim 9, wherein said neutral group is an oxyalkylene.
- 13. A compound comprising an oligonucleotide and in any order a mass-modifying region, a charge-modifying region and a detectable region joined by phosphate linkages.
- 14. A compound according to claim 13, where said phosphate linkages comprise said charge-modifying region and said neutral region is an alkylene, an alkyleneoxy or a substituted aromatic group.
- 15. A compound according to claim 13, wherein said detectable region is a fluorescer.
- 16. A compound according to claim 15, wherein said fluorescer is fluorescein, a fluorescein derivative, rhodamine, a rhodamine derivative, Cy-3 or Cy-5.
- 17. A method of performing a multiplexed assay for the determination of a plurality of target species in a sample employing eTag reporter conjugated binding compounds, wherein said eTag reporters are linked to said binding compounds by a cleavable linkage, are specific for the binding compound to which said eTag reporter is conjugated, are other than oligonucleotides of at least 3 nucleotides and have at least one characteristic for individual detection, and said binding compounds are individually specific for different target species, said method comprising:
combining said sample with said compounds for said target species under conditions for binding of said binding compounds to said target species; releasing eTag reporters from eTag reporter conjugated binding compounds bound to said target species by cleavage of said cleavable linkage; and identifying said released eTag reporters by means of said at least one characteristic; whereby the presence of said target compounds in said sample is determined.
- 18. A method according to claim 17, wherein said target species are nucleic acid sequences homologous to said binding compounds.
- 19. A method according to claim 18, wherein said releasing is by cleavage of a phosphate bond.
- 20. A method according to claim 17, wherein said target species are poly(amino acids).
- 21. A method according to claim 20, wherein said cleavable linkage is photolytically labile.
- 22. A method according to claim 20, wherein said releasing results from an active agent in proximity to said cleavable linkage.
- 23. A method according to claim 17, wherein said at least one characteristic is mobility in an electrical field.
- 24. A method according to claim 17, wherein said at least one characteristic is mass for identification in a mass spectrometer.
- 25. A method of performing a multiplexed assay for the determination of a plurality of nucleic acid target species in a sample employing eTag reporter substituted oligonucleotides, wherein said oligonucleotides are homologous to said target species and wherein said eTag reporters are specific for said oligonucleotides to which said eTag reporter is substituted, are other than oligonucleotides of at least 3 nucleotides and have at least one characteristic for individual detection, said method comprising:
combining said sample with said oligonucleotides for said target species under conditions for hybridization of said oligonucleotides to said target species; releasing eTag reporters from eTag reporter conjugated oligonucleotides bound to said target species by cleavage of a phosphate bond of said oligonucleotide; and identifying said released eTag reporters by means of said at least one characteristic; whereby the presence of said target compounds in said sample is determined.
- 26. A method according to claim 25, wherein said cleavage of said phosphate bond releases said eTag reporter comprising one nucleotide.
- 27. A method according to claim 25, wherein said releasing includes a cleavase.
- 28. A method according to claim 25, wherein said releasing includes a polymerase having nuclease activity.
- 29. A method according to claim 25, wherein said releasing comprises as an additional step adding a primer to said sample homologous to a first portion of said target species 5′ of a second portion of said target species to which said oligonucleotide is homologous.
- 30. A method according to claim 25, wherein said identifying comprises separating said released eTag reporters in an electrical field.
- 31. A method according to claim 25, wherein said identifying comprises separating said released eTag reporters in a magnetic field.
- 32. A method of performing a multiplexed assay for the determination of a plurality of poly(amino acid) target species in a sample employing eTag reporter conjugated binding compounds, wherein said eTag reporters are linked to said binding compounds by a cleavable linkage, are specific for the binding compound to which said eTag reporter is conjugated, are other than oligonucleotides of at least 3 nucleotides and have at least one characteristic for individual detection, and said binding compounds are individually specific for different target species, said method comprising:
combining said sample with said compounds for said target species under conditions for binding of said binding compounds to said target species; releasing eTag reporters from eTag reporter conjugated binding compounds bound to said target species by cleavage of said cleavable linkage; and identifying said released eTag reporters by means of said at least one characteristic; whereby the presence of said target compounds in said sample is determined.
- 33. A method according to claim 32, wherein said cleavable linkage is photolytically labile.
- 34. A method according to claim 32, wherein said releasing comprises bringing said cleavable linkage in proximity to an active agent.
- 35. A method according to claim 34, wherein said active agent is singlet oxygen.
- 36. A method according to claim 34, wherein said active agent is produced at a solid support.
- 37. A method according to claim 34, wherein said active agent is produced by a second binding compound bound to said target species.
- 38. A method according to claim 32, wherein said binding compounds are antibodies or fragments thereof.
- 39. A method according to claim 32. wherein said identifying comprises separation in an, electric field.
- 40. A method according to claim 32, wherein said separation comprises separation in a magnetic field.
- 41. A method of performing a multiplexed assay for the determination of a plurality of target species in a sample employing eTag reporter conjugated binding compounds, wherein said eTag reporters are linked to said binding compounds by a cleavable linkage, are specific for the binding compound to which said eTag reporter is conjugated, are other than oligonucleotides of at least 3 nucleotides and have at least one characteristic for individual detection, and wherein said eTag reporter conjugated binding compound comprises a ligand bound at a site where said ligand remains with said binding compound upon cleavage of said cleavable linkage, said method comprising:
combining said sample with said compounds for said target species under conditions for binding of said binding compounds to said target species; releasing eTag reporters from eTag reporter conjugated binding compounds bound to said target species by cleavage of said cleavable linkage; adding a reciprocal binding member to said binding compound to bind to said eTag reporter conjugated binding compounds to reduce interfence with said determination; and identifying said released eTag reporters by means of said at least one characteristic; whereby the presence of said target compounds in said sample is determined.
- 42. A method according to claim 41, wherein said identifying is by separating in an electrical field and said reciprocal binding member has the opposite polarity of said eTag reporters.
- 43. A method according to claim 42, wherein said eTag reporters are negatively charged and said reciprocal binding member is positively charged.
- 44. A method according to claim 43, wherein said ligand is biotin and said reciprocal binding member is streptavidin.
- 45. A method for determining the change in the surface membrane protein population for a plurality of surface membrane proteins by performing a multiplexed assay for the determination of said plurality of surface membrane proteins of at least one cell in a cellular sample by employing eTag reporter conjugated binding compounds, wherein said eTag reporters are linked to said binding compounds by a cleavable linkage, are specific for the binding compound to which said eTag reporter is conjugated, are other than oligonucleotides of at least 3 nucleotides and have at least one characteristic for individual detection, said method comprising:
combining said cellular sample with said compounds for said proteins under conditions for binding of said binding compounds to said proteins; releasing eTag reporters from eTag reporter conjugated binding compounds bound to said proteins by cleavage of said cleavable linkage; and identifying said released eTag reporters by means of said at least one characteristic; whereby the presence of said proteins in said sample is determined.
- 46. A method according to claim 45, wherein said binding compounds consist of at least one of ligands for said surface membrane proteins and antibodies to said surface membrane proteins.
- 47. A method according to claim 45, wherein said combining includes the addition of second binding compounds conjugated with an active agent producing moiety, wherein said active agent causes cleavage of said cleavable linkage.
- 48. A method according to claim 47, wherein said active agent is singlet oxygen.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuing patent application of application Ser. No. 09/602,586, filed Jun. 21, 2000, which is a continuing application of application Ser. No. 09/561,579, filed Apr. 28, 2000.
Divisions (1)
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Number |
Date |
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Parent |
09698846 |
Oct 2000 |
US |
Child |
10420549 |
Apr 2003 |
US |
Continuations (2)
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09602586 |
Jun 2000 |
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09698846 |
Oct 2000 |
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Parent |
09561579 |
Apr 2000 |
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09602586 |
Jun 2000 |
US |