Tag library compounds, compositions, kits and methods of use
First Claim
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.
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Abstract
Families of compositions are provided as labels, referred to as eTag reporters for attaching to polymeric compounds and assaying based on release of the eTag reporters from the polymeric compound and separation and detection. For oligonucleotides, the eTag reporters are synthesized at the end of the oligonucleotide by using phosphite or phosphate chemistry, whereby mass-modifying regions, charge-modifying regions and detectable regions are added sequentially to produce the eTag labeled reporters. By using small building blocks and varying their combination large numbers of different eTag reporters can be readily produced attached to a binding compound specific for the target compound of interest for identification. Protocols are used that release the eTag reporter when the target compound is present in the sample.
86 Citations
48 Claims
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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:
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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. - View Dependent Claims (2, 3)
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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:
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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 - View Dependent Claims (5, 6, 7, 8)
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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:
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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. - View Dependent Claims (10, 11, 12)
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- 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.
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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:
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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. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24)
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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:
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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. - View Dependent Claims (26, 27, 28, 29, 30, 31)
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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:
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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. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40)
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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:
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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. - View Dependent Claims (42, 43, 44)
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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:
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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. - View Dependent Claims (46, 47, 48)
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Specification