Protein expression profiling
First Claim
1. A method for detecting one or more analytes, the method comprising (a) bringing into contact one or more analyte samples and one or more arrays, wherein each array comprises a set of analyte capture agents, wherein each analyte capture agent is immobilized on a solid support in a different predefined region of the solid support, wherein each analyte capture agent interacts with an analyte directly or indirectly, (b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, (c) simultaneous with, or following, either or both steps (a) and (b), incubating the analyte samples, the arrays, and the reporter binding primers under conditions that promote interaction of the specific binding molecules, analytes, and analyte capture agents, (d) prior to, simultaneous with, or following step (b), bringing into contact the reporter binding primers, one or more first-stage amplification target circles, one or more second-stage primers, and one or more second-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, wherein the second-stage primers each comprise a first portion and a second portion, wherein the second-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the second portion of at least one of the second-stage primers, and incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers and between the second-stage primers and the second-stage amplification target circles, (e) following step (d) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein each second-stage primer can interact with tandem sequence DNA produced from at least one of the first-stage amplification target circles, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.
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Abstract
Disclosed are compositions and methods for detecting small quantities of analytes such as proteins and peptides. The method involves associating a primer with an analyte and subsequently using the primer to mediate rolling circle replication of a circular DNA molecule. Amplification of the DNA circle is dependent on the presence of the primer. Thus, the disclosed method produces an amplified signal, via rolling circle amplification, from any analyte of interest. The amplified DNA remains associated with the analyte, via the primer, and so allows spatial detection of the analyte. The disclosed method can be used to detect and analyze proteins and peptides. Multiple proteins can be analyzed using microarrays to which the various proteins are immobilized. A rolling circle replication primer is then associated with the various proteins using a conjugate of the primer and a molecule that specifically binds the proteins to be detectable. Rolling circle replication from the primers results in production of a large amount of DNA at the sites in the array where the proteins are immobilized. The DNA produced by rolling circle replication can be further amplified in secondary and higher order amplification processes using second-stage or higher order primers in conjunction with second-stage or higher order amplification target circles. The amplified DNA serves as a readily detectable signal for the proteins. The disclosed method can also be used to compare the proteins expressed in two or more different samples. The information generated is analogous to the type of information gathered in nucleic acid expression profiles. The disclosed method allows sensitive and accurate detection and quantitation of proteins expressed in any cell or tissue.
115 Citations
64 Claims
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1. A method for detecting one or more analytes, the method comprising
(a) bringing into contact one or more analyte samples and one or more arrays, wherein each array comprises a set of analyte capture agents, wherein each analyte capture agent is immobilized on a solid support in a different predefined region of the solid support, wherein each analyte capture agent interacts with an analyte directly or indirectly, (b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, (c) simultaneous with, or following, either or both steps (a) and (b), incubating the analyte samples, the arrays, and the reporter binding primers under conditions that promote interaction of the specific binding molecules, analytes, and analyte capture agents, (d) prior to, simultaneous with, or following step (b), bringing into contact the reporter binding primers, one or more first-stage amplification target circles, one or more second-stage primers, and one or more second-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, wherein the second-stage primers each comprise a first portion and a second portion, wherein the second-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the second portion of at least one of the second-stage primers, and incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers and between the second-stage primers and the second-stage amplification target circles, (e) following step (d) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein each second-stage primer can interact with tandem sequence DNA produced from at least one of the first-stage amplification target circles, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.
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50. A method for detecting one or more analytes, the method comprising
(a) bringing into contact one or more analyte samples and one or more arrays, wherein each array comprises a set of analyte capture agents, wherein each analyte capture agent is immobilized on a solid support in a different predefined region of the solid support, wherein each analyte capture agent interacts with an analyte directly or indirectly, (b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, (c) simultaneous with, or following, either or both steps (a) and (b), incubating the analyte samples, the arrays, and the reporter binding primers under conditions that promote interaction of the specific binding molecules, analytes, and analyte capture agents, (d) prior to, simultaneous with, or following step (b), bringing into contact the reporter binding primers and one or more first-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and incubating the reporter binding primers and first-stage amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers, (e) following step (d) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the reporter binding primers and first-stage amplification target circles under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of primary tandem sequence DNA, (f) following step (e) and prior to, simultaneous with, or following steps (a), (b), or (c), bringing into contact the primary tandem sequence DNA and one or more second-stage primers, wherein second-stage primer each comprise a first portion and a second portion, wherein the first portion can interact with the primary tandem sequence DNA, wherein the second portion is not complementary to the primary tandem sequence DNA, and incubating the primary tandem sequence DNA and the second-stage primers under conditions that promote hybridization of the first portion of the second-stage primers to the primary tandem sequence DNA, (g) following step (f) and prior to, simultaneous with, or following steps (a), (b), or (c), bringing into contact the second-stage primers and one or more second-stage amplification target circles, and incubating under conditions promoting hybridization of the second-stage amplification target circles and the second portion of the second-stage primers, (h) following step (g) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the second-stage primers and the second-stage amplification target circles under conditions that promote replication of the second-stage amplification target circles, wherein replication of the second-stage amplification target circles results in formation of secondary tandem sequence DNA, wherein detection of primary tandem sequence DNA, secondary tandem sequence DNA or both indicates the presence of the corresponding analytes.
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51. A method for detecting one or more analytes, the method comprising
(a) bringing into contact one or more analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, and incubating the analyte samples and the reporter binding primers under conditions that promote interaction of the specific binding molecules and analytes, (b) prior to, simultaneous with, or following step (a), bringing into contact one or more first analyte capture agents and one or more first analyte samples, and bringing into contact one or more second analyte capture agents and one or more second analyte samples, wherein each analyte capture agent comprises an analyte interaction portion and a capture portion, wherein for each first analyte capture agent there is a matching second analyte capture agent, wherein the analyte interaction portions of the first analyte capture agents interact with the same analyte as the analyte interaction portions of the matching second analyte capture agents, wherein the capture portions of the first and second analyte capture agents each interact with a specific binding molecule of one or more of the reporter binding primers, wherein the capture portions of the first analyte capture agents interact with different specific binding molecules than the capture portions of the matching second analyte capture agents, (c) prior to, simultaneous with, or following step (a), bringing into contact the reporter binding primers, one or more first-stage amplification target circles, one or more second-stage primers, and one or more second-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, wherein the second-stage primers each comprise a first portion and a second portion, wherein the second-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the second portion of at least one of the second-stage primers, and incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers and between the second-stage primers and the second-stage amplification target circles, (d) following step (c) and prior to, simultaneous with, or following step (a), incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote replication of the amplification target circles, wherein each different specific binding molecule is part of a different one of the reporter binding primers, wherein the rolling circle replication primer of each different reporter binding primer is different, wherein each different rolling circle replication primer primes replication of a different one of the first-stage amplification target circles, wherein each different first-stage amplification target circle produces a different tandem sequence DNA, wherein each second-stage primer is different, wherein the first portion of each different second-stage primer matches sequence in a different one or the first-stage amplification target circle, wherein each different second-stage primer primes replication of a different one of the second-stage amplification target circles, wherein each different second-stage amplification target circle produces a different tandem sequence DNA, wherein each second-stage primer can interact with tandem sequence DNA produced from at least one of the first-stage amplification target circles, wherein the presence or absence of the same analyte in different analyte samples is indicated by the presence or absence of corresponding tandem sequence DNA.
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57. A method for detecting one or more analytes, the method comprising
(a) bringing into contact one or more analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, and incubating the analyte samples and the reporter binding primers under conditions that promote interaction of the specific binding molecules and analytes, (b) prior to, simultaneous with, or following step (a), bringing into contact one or more first analyte capture agents and one or more first analyte samples, and bringing into contact one or more second analyte capture agents and one or more second analyte samples, wherein each analyte capture agent comprises an analyte interaction portion and a capture portion, wherein for each first analyte capture agent there is a matching second analyte capture agent, wherein the analyte interaction portions of the first analyte capture agents interact with the same analyte as the analyte interaction portions of the matching second analyte capture agents, wherein the capture portions of the first and second analyte capture agents each interact with a specific binding molecule of one or more of the reporter binding primers, wherein the capture portions of the first analyte capture agents interact with different specific binding molecules than the capture portions of the matching second analyte capture agents, (c) prior to, simultaneous with, or following step (a), bringing into contact the reporter binding primers and one or more first-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and incubating the reporter binding primers and first-stage amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers, (d) following step (c) and prior to, simultaneous with, or following step (a), incubating the reporter binding primers and first-stage amplification target circles under conditions that promote replication of the first-stage amplification target circles, wherein replication of the first-stage amplification target circles results in the formation of primary tandem sequence DNA, (e) following step (d) and prior to, simultaneous with, or following step (a), bringing into contact the primary tandem sequence DNA and one or more second-stage primers, wherein second-stage primer each comprise a first portion and a second portion, wherein the first portion can interact with the primary tandem sequence DNA, wherein the second portion is not complementary to the primary tandem sequence DNA, and incubating the primary tandem sequence DNA and the second-stage primers under conditions that promote hybridization of the first portion of the second-stage primers to the primary tandem sequence DNA, (f) following step (e) and prior to, simultaneous with, or following step (a), bringing into contact the second-stage primers and one or more second-stage amplification target circles, and incubating under conditions promoting hybridization of the second-stage amplification target circles and the second portion of the second-stage primers, (g) following step (f) and prior to, simultaneous with, or following step (a), incubating the second-stage primers and the second-stage amplification target circles under conditions that promote replication of the second-stage amplification target circles, wherein replication of the second-stage amplification target circles results in formation of secondary tandem sequence DNA, wherein each different specific binding molecule is part of a different one of the reporter binding primers, wherein the rolling circle replication primer of each different reporter binding primer is different, wherein each different rolling circle replication primer primes replication of a different one of the first-stage amplification target circles, wherein each different first-stage amplification target circle produces a different tandem sequence DNA, wherein each second-stage primer is different, wherein the first portion of each different second-stage primer matches sequence in a different one or the first-stage amplification target circle, wherein each different second-stage primer primes replication of a different one of the second-stage amplification target circles, wherein each different second-stage amplification target circle produces a different tandem sequence DNA, wherein the presence or absence of the same analyte in different analyte samples is indicated by the presence or absence of corresponding primary tandem sequence DNA, secondary tandem sequence DNA, or both.
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58. A method for detecting one or more analytes, the method comprising
(a) treating one or more analyte samples so that one or more analytes are modified, (b) bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with a modified analyte directly or indirectly, and incubating the analyte samples and the reporter binding primers under conditions that promote interaction of the specific binding molecules and modified analytes, (c) prior to, simultaneous with, or following steps (a) or (b), bringing into contact the reporter binding primers, one or more first-stage amplification target circles, one or more second-stage primers, and one or more second-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, wherein the second-stage primers each comprise a first portion and a second portion, wherein the second-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the second portion of at least one of the second-stage primers, and incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers and between the second-stage primers and the second-stage amplification target circles, (d) following step (c) and prior to, simultaneous with, or following steps (a) or (b), incubating the reporter binding primers, second-stage primers, and amplification target circles under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein each second-stage primer can interact with tandem sequence DNA produced from at least one of the first-stage amplification target circles, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.
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60. A method for detecting one or more analytes, the method comprising
(a) treating one or more analyte samples so that one or more analytes are modified, (b) bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with a modified analyte directly or indirectly, and incubating the analyte samples and the reporter binding primers under conditions that promote interaction of the specific binding molecules and modified analytes, (c) prior to, simultaneous with, or following steps (a) or (b), bringing into contact the reporter binding primers and one or more first-stage amplification target circles, wherein the first-stage amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and incubating the reporter binding primers and first-stage amplification target circles under conditions that promote hybridization between the first-stage amplification target circles and the rolling circle replication primers, (d) following step (c) and prior to, simultaneous with, or following steps (a) or (b), incubating the reporter binding primers and first-stage amplification target circles under conditions that promote replication of the first-stage amplification target circles, wherein replication of the first-stage amplification target circles results in the formation of primary tandem sequence DNA, (e) following step (d) and prior to, simultaneous with, or following steps (a) or (b), bringing into contact the primary tandem sequence DNA and one or more second-stage primers, wherein second-stage primer each comprise a first portion and a second portion, wherein the first portion can interact with the primary tandem sequence DNA, wherein the second portion is not complementary to the primary tandem sequence DNA, and incubating the primary tandem sequence DNA and the second-stage primers under conditions that promote hybridization of the first portion of the second-stage primers to the primary tandem sequence DNA, (f) following step (e) and prior to, simultaneous with, or following steps (a) or (b), bringing into contact the second-stage primers and one or more second-stage amplification target circles, and incubating under conditions promoting hybridization of the second-stage amplification target circles and the second portion of the second-stage primers, (g) following step (f) and prior to, simultaneous with, or following steps (a) or (b), incubating the second-stage primers and the second-stage amplification target circles under conditions that promote replication of the second-stage amplification target circles, wherein replication of the second-stage amplification target circles results in formation of secondary tandem sequence DNA, wherein detection of primary tandem sequence DNA, secondary tandem sequence DNA or both indicates the presence of the corresponding analytes.
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61. A method for detecting one or more analytes, the method comprising
(a) bringing into contact one or more analyte samples and one or more arrays, wherein each array comprises a set of analyte capture agents, wherein each analyte capture agent is immobilized on a solid support in a different predefined region of the solid support, wherein each analyte capture agent interacts with an analyte directly or indirectly, (b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, (c) simultaneous with, or following, either or both steps (a) and (b), incubating the analyte samples, the arrays, and the reporter binding primers under conditions that promote interaction of the specific binding molecules, analytes, and analyte capture agents, (d) prior to, simultaneous with, or following step (b), bringing into contact the reporter binding primers, one or more amplification target circles, and one or more secondary DNA strand displacement primers, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, wherein the secondary DNA strand displacement primers each comprise a matching portion, wherein the matching portion matches sequence of at least one of the amplification target circles, and incubating the reporter binding primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers, (e) following step (d) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the reporter binding primers and amplification target circles under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.
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63. A method for detecting one or more analytes, the method comprising
(a) bringing into contact one or more analyte samples and one or more arrays, wherein each array comprises a set of analyte capture agents, wherein each analyte capture agent is immobilized on a solid support in a different predefined region of the solid support, wherein each analyte capture agent interacts with an analyte directly or indirectly, (b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, (c) simultaneous with, or following, either or both steps (a) and (b), incubating the analyte samples, the arrays, and the reporter binding primers under conditions that promote interaction of the specific binding molecules, analytes, and analyte capture agents, (d) prior to, simultaneous with, or following step (b), bringing into contact the reporter binding primers and one or more amplification target circles, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and incubating the reporter binding primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers, (e) following step (d) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the reporter binding primers and amplification target circles under conditions that promote exponential rolling circle amplification, wherein exponential rolling circle amplification results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.
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64. A method comprising
(a) bringing into contact one or more analyte samples and one or more arrays, wherein each array comprises a set of analyte capture agents, wherein each analyte capture agent is immobilized on a solid support in a different predefined region of the solid support, wherein each analyte capture agent interacts with an analyte directly or indirectly, (b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding primers, wherein each reporter binding primer comprises a specific binding molecule and a rolling circle replication primer, wherein each specific binding molecule interacts with an analyte directly or indirectly, (c) simultaneous with, or following, either or both steps (a) and (b), incubating the analyte samples, the arrays, and the reporter binding primers under conditions that promote interaction of the specific binding molecules, analytes, and analyte capture agents, (d) prior to, simultaneous with, or following step (b), bringing into contact the reporter binding primers and one or more amplification target circles, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and incubating the reporter binding primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers, (e) following step (d) and prior to, simultaneous with, or following steps (a), (b), or (c), incubating the reporter binding primers and amplification target circles under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA.
Specification