Systems, tools and methods of assaying biological materials using spatially-addressable arrays
First Claim
1. An assay system for multiplexing on a single array one or more biological samples having one or more biological targets per sample, comprising:
- an array apparatus that comprises a first plurality of biological probes on a substrate in an array pattern of features, wherein each first probe of the first plurality is different from others in the first plurality, each different first probe is located at a different feature location of the array and is a different address on the array; and
a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region, wherein the first region of each second probe is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes.
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Abstract
Systems, tools and methods of assaying biological material are used to perform complex sandwich hybridization assays. The tools used comprise biological solution probes that are customized for each assay. The solution probe comprises a first region for hybridizing to a probe, in a generic set of capture probes on a universal assay apparatus, and a second region for hybridizing to a target in a sample. The solution probe assembles the target to the assay apparatus by hybridizing the second region to the target and the first region to the capture probe. In array assays, one or more biological samples, having one or more targets per sample, can be multiplexed on the same universal array comprising the generic set of capture probes in an array pattern of features on the substrate. The customized solution probe addresses and assembles a predetermined target-sample combination onto the array at a corresponding capture probe address location. The systems, tools and methods have specificity and sensitivity by systematically providing a reduced likelihood of cross-hybridizations and intramolecular structures in the probes. Specificity and sensitivity of the assay are provided by the incorporation of a chemically modified monomer in the capture probe and a similarly modified monomer complement in the first region of the solution probe. The modified monomers preferentially hybridize with each other. When the probe and respective probe region are oligonucleotides, the complementary modified nucleotides have a reversed polarity relative to the polarity of the respective probe and probe region. The complementary reversed polarity nucleotides form a thermodynamically more stable hybridization to each other than a hybridization between the reversed polarity nucleotide and a complementary nucleotide whose polarity is not similarly reversed.
17 Citations
100 Claims
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1. An assay system for multiplexing on a single array one or more biological samples having one or more biological targets per sample, comprising:
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an array apparatus that comprises a first plurality of biological probes on a substrate in an array pattern of features, wherein each first probe of the first plurality is different from others in the first plurality, each different first probe is located at a different feature location of the array and is a different address on the array; and
a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region, wherein the first region of each second probe is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 86, 87, 88, 96, 97, 100)
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13. An assay method of multiplexing on a single array one or more biological samples having one or more biological targets per sample, comprising the steps of:
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providing an array apparatus having a first plurality of biological probes on a substrate in an array pattern of features, each first probe of the first plurality being different, each different first probe being located on a different feature location of the array, wherein each different first probe is a different address on the array apparatus;
providing a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region that is complementary to a different one of the first probes, the second region of each second probe being complementary to a target from the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes;
assembling the different target-sample combinations to the array with the second set of probes;
removing unassembled biological materials from the array; and
analyzing assay results comprising the step of determining the presence of the one or more targets in the one or more samples from whether the different target-sample combinations are assembled on the array at the corresponding first probe locations.
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22. A sandwich hybridization system for assaying biological material having specificity and sensitivity comprising:
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an apparatus having a first plurality of biological probes attached to a substrate, each first probe of the first plurality comprising a first sequence of monomers, wherein at least one first monomer of the first sequence is chemically modified; and
a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, the first region comprising a second sequence of monomers, wherein at least one second monomer is modified similarly to the first modified monomer, the second sequence of monomers and the second modified monomer are respectively complementary to the first sequence of monomers and first modified monomer in the first probe, and wherein the second region is complementary to the biological material to be assayed, wherein the first modified monomer preferentially hybridizes to the complementary second modified monomer during the assay instead of to a complementary monomer that has not been similarly modified. - View Dependent Claims (23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 89)
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28. A sandwich hybridization system for assaying biological material having specificity and sensitivity comprising:
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an apparatus having a first plurality of oligonucleotide probes attached to a substrate, each first probe of the first plurality comprising;
a first sequence of nucleotides having either a (3′
→
5′
) or (5′
→
3′
) polarity,at least one first nucleotide that has a reversed polarity relative to the polarity of the first sequence; and
a second plurality of biological probes, each second probe of the second plurality comprising a first oligonucleotide region and a second region, the first oligonucleotide region comprising;
a second sequence of nucleotides having the same polarity as the polarity of the the first sequence, and are respectively complementary to the first sequence of nucleotides, and at least one second nucleotide that is complementary to the at least one reversed polarity first nucleotide, the second nucleotide having a reversed polarity relative to the polarity of the second sequence; and
the second region is complementary to the biological material to be assayed, and wherein the reversed polarity first nucleotide and the complementary reversed polarity second nucleotide preferentially hybridizes to each other during the assay than to a complementary nucleotide whose polarity is not similarly reversed.
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37. A sandwich hybridization method of assaying biological materials with specificity and sensitivity comprising the steps of:
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providing an apparatus having a first set of biological probes on a surface of a substrate, each first probe of the first set comprising a sequence of monomers, wherein at least one first monomer of the first sequence is chemically modified;
providing a second set of biological probes, each second probe of the second set comprises a first region and a second region, the first region of each second probe comprises a second sequence of monomers, wherein at least one second monomer of the second sequence is chemically modified similarly to the first modified monomer, the second sequence of monomers and second modified monomer being complementary respectively to the first sequence and first modified monomer, and wherein the second region of each second probe is complementary to a biological material to be assayed, hybridizing the biological material to the second set of probes and hybridizing the second set of probes to the first set of probes on the apparatus, wherein the first modified monomer preferentially hybridizes to the complementary second modified monomer instead of to a complementary monomer that has not been similarly modified;
removing unhybridized biological material from the apparatus; and
analyzing assay results.
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40. A assay system with specificity and sensitivity for multiplexing one or more biological samples, having one or more biological targets per sample on a single array comprising:
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an array apparatus that comprises a first plurality of biological probes on a substrate in an array pattern of features, wherein each first probe of the first plurality comprises a different first sequence of monomers, wherein at least one first monomer of the first sequence comprises a chemical modification, and wherein each different first probe is located at a different feature location of the array and is a different address on the array apparatus; and
a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region, wherein each different first region comprises a second sequence of monomers, the second sequence comprising at least one second monomer having a chemical modification similar to, and being complementary to, the first modified monomer, wherein each different first region is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes , and wherein the first modified monomer and the second modified monomer preferentially hybridize together instead of to a monomer that has not been similarly modified. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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51. An assay method of multiplexing on a single array one or more biological samples having one or more biological targets per sample, the method having specificity and sensitivity and comprising the steps of:
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providing an array apparatus having a first plurality of biological probes on a substrate in an array pattern of features, each first probe of the plurality being different from others in the first plurality by comprising a different first sequence of monomers, wherein the first sequence of monomers comprises at least one chemically modified monomer, each different first probe being located on a different feature location of the array apparatus, wherein each different first probe -is a different address on the array apparatus;
providing a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region that is complementary to a different one of the first probes, the first region comprising a second sequence of monomers, wherein the second sequence of monomers comprises at least one monomer similarly modified as and being complementary to the first modified monomer, the second region of each second probe being complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes;
assembling the different target-sample combinations to the array with the second set of probes, comprising the steps of;
incubating the one or more biological samples with the second plurality of probes to hybridize the biological targets per sample with complementary second regions of the second probes; and
incubating the second plurality of probes with the first plurality of probes on the array apparatus to preferentially hybridize the first regions of the second plurality of probes to corresponding complementary first probes by hybridizing the second modified monomer of each first region to its complementary first modified monomer of each first probe instead of with a complementary monomer that has not been similarly modified;
removing unassembled biological materials from the array; and
analyzing assay results comprising the step of determining the presence of the one or more targets in the one or more samples from whether the different target-sample combinations are assembled on the array at the corresponding first probe locations. - View Dependent Claims (52, 90)
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53. A assay system with specificity and sensitivity for multiplexing one or more biological samples, having one or more biological targets per sample on a single array comprising:
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an array apparatus that comprises a first plurality of oligonucleotide probes on a substrate in an array pattern of features, wherein each first probe of the first plurality comprises;
a first sequence of nucleotides having either a (3′
→
5′
) or (5′
→
3′
) polarity, at least one first nucleotide has a reversed polarity relative to the polarity of the first sequence, andwherein each first sequence is different from other first sequences, each different first probe is located at a different feature location of the array, and each different first probe is a different address on the array apparatus; and
a second plurality of biological probes, each second probe of the second plurality comprising an oligonucleotide first region and a second region, each second probe being different from others in the second plurality by comprising a different oligonucleotide first region complementary to a different one of the first oligonucleotide probes, wherein each different oligonucleotide first region comprises;
a second sequence of nucleotides having the same polarity as the polarity of the first plurality of probes, at least one second nucleotide having a reversed polarity relative to the polarity of the second sequence, and wherein the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes, wherein the first reversed polarity nucleotide and the second reversed polarity nucleotide preferentially hybridize to each other by forming a more thermodynamically stable hybridization than to a complementary nucleotide whose polarity is not similarly reversed. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 94, 98, 99)
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69. An assay method of multiplexing on a single array one or more biological samples having one or more biological targets per sample, the method having specificity and sensitivity and comprising the steps of:
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providing an array apparatus having a first plurality of biological probes on a substrate in an array pattern of features, each first probe of the plurality being a different oligonucleotide by comprising a different first sequence of nucleotides, wherein the first sequence of nucleotides having either a (3′
→
5′
) or (5′
→
3′
) polarity, at least one first nucleotide has a reversed polarity relative to the polarity of the first sequence, and wherein each different first probe is located at a different feature location of the array, and each different first probe is a different address on the array apparatus;
providing a second set of biological probes, each second probe of the second set comprising an oligonucleotide first region and a second region, each second probe being different from others in the second set by comprising a different oligonucleotide first region complementary to a different one of the first oligonucleotide probes, each different oligonucleotide first region comprises a different second sequence of nucleotides having the same polarity as the first sequence of nucleotides, at least one second nucleotide having a reversed polarity relative to the polarity of the second sequence, and wherein the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes;
assembling the different target-sample combinations to the array with the second set of probes;
removing unassembled biological materials from the array; and
analyzing assay results comprising the step of determining the presence of the one or more targets in the one or more samples from whether the different target-sample combinations are assembled on the array at the corresponding first probe locations.
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77. A kit for multiplexing one or more biological samples having one or more targets per sample in an addressable, self-assembling assay on a single array comprising:
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an array apparatus that comprises a first set of biological probes on a substrate in an array pattern of features, wherein each first probe of the first set is different from others in the first set, each different first probe is located at a different feature location of the array corresponding to a different address on the array;
a second set of biological probes, each second probe of the second set comprising a first region and a second region, each second probe being different from others in the second set by comprising a different first region, wherein the first region of each second probe is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different predetermined combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes ; and
instructions for using the apparatus and second set of probes in a multiplexing assay of target-sample combinations selected from one target per sample in a plurality of different samples, a plurality of different targets per sample in one sample, or a plurality of different targets per sample in a plurality of different samples. - View Dependent Claims (78, 79)
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80. A set of biological probes in solution for sandwich hybridization assays of one or more biological targets from one or more biological samples multiplexed on an array of biological features, the set of biological probes comprising a plurality of different solution probes, each different solution probe comprises:
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a different first probe region complementary to a different one of the biological features on the array; and
a second probe region complementary to a biological target in the one or more samples, such that during an assay, the set of solution probes assembles the one or more biological targets from the one or more biological samples to different biological features on the array. - View Dependent Claims (81, 82, 83, 84, 85)
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95. An array with specificity and sensitivity for multiplexing one or more biological samples, having one or more biological targets per sample comprising:
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a substrate with a surface; and
a plurality of oligonucleotide probes on the surface of the substrate in an array pattern of features, wherein the oligonucleotide probes of the plurality are all different from each other, each different probe of the plurality comprises;
a sequence of nucleotides having either a (3′
→
5′
) or (5′
→
3′
) polarity, andat least one nucleotide having a reversed polarity relative to the polarity of the sequence, wherein the sequence for each different probe is different, each different first probe is located at a different feature location of the array, and each different first probe is a different address for a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed.
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Specification