Multiplex method for detecting different analytes in a sample
The technology provided herein relates to multiplex methods and kits for detecting different analytes in a sample in parallel by sequential signal-encoding of said analytes, as well as in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.
1 . A method of assigning coded fluorescence patterns to a plurality of target analytes in a sample, comprising: subjecting the sample to a plurality of detection rounds, each detection round comprising: contacting the sample to a decoding oligonucleotide probe population comprising decoding oligonucleotide probes that each recruit, by reverse complementary base pairing, one translator connector element comprising a fluorescence moiety signal element selected from at least two distinct populations of fluorescence moieties to at least some of the plurality of target analytes; wherein the decoding oligonucleotide probes do not directly bind target analytes; and wherein the decoding oligonucleotide probes are recruited to their target analytes by analyte specific probes, each analyte specific probe having an identifier element that is reverse complementary to a portion of at least one decoding oligonucleotide; contacting the sample to the at least two populations of translator connector elements differing in their fluorescence moieties, wherein the at least two populations of translator connector element differ in fluorescence moiety from one another; assaying for fluorescence in the sample; removing the decoding oligonucleotide probe population and the translator connector elements after each round of the assaying without removing the analyte specific probes, by heating the sample until base pairings in the decoding oligonucleotide probe population are destabilized but base pairings between the analyte specific probes and the analytes remain intact; wherein the decoding oligonucleotide probes of the decoding oligonucleotide probe population vary across the plurality of detection rounds causing the translator connector element comprising a fluorescence moiety signal element recruited to a target analyte to vary in fluorescence moiety across the plurality of detection rounds; and wherein the translator connector elements differing in their fluorescence moieties do not vary in composition across the plurality of detection rounds; wherein a fluorescence pattern at an analyte position in the sample is specified by order of decoding oligonucleotide probe addition to the sample.
2 . The method of claim 1 , wherein a number of patterns detectable increases exponentially with a number of detection rounds.
3 . The method of claim 1 , wherein the translator connector elements do not comprise nucleic acid tags that are specific to the target analytes.
4 . The method of claim 1 , wherein separate aliquots of common translator connector elements are used across multiple detection rounds.
5 . The method of claim 1 , wherein the plurality of detection rounds comprises at least 5 detection rounds.
6 . The method of claim 1 , wherein the method comprises use of no more than two populations of translator connector elements.
7 . The method of claim 1 , wherein the analyte specific probes comprise oligo-tagged antibodies.
8 . The method of claim 1 , wherein the analyte specific probes comprise binding element portions that are reverse complementary to adjacent regions of a nucleotide analyte target, and identifier element portions that identify the target analyte for binding by at least some of the decoding oligonucleotide probes.
9 . The method of claim 1 , wherein a signal element fluorescence moiety of the translator connector elements is reverse complementary to a portion of a subset of the decoding oligonucleotide probes, causing members of a population of the translator connector elements sharing a common fluorescence moiety to be recruited to a plurality of target analytes tagged by distinct analyte specific probes differing in their identifier elements.
10 . The method of claim 9 , wherein members of a population of the translator connector elements do not specifically bind an analyte in the absence of a decoding oligonucleotide probe.
11 . The method of claim 1 , wherein the order of delivery of decoding oligonucleotide probes that bind an analyte specific probe identifier element assigns a coded fluorescence pattern to the target analyte that specifically identifies the analyte.
12 . The method of claim 1 , wherein the order of delivery decoding oligonucleotide probes that bind an analyte specific probe identifier element assigns a coded fluorescence pattern to the target analyte that uniquely identifies the analyte.
13 . The method of claim 1 , wherein the coded fluorescence pattern is not determined by a linear series of binding sites on the analyte.
14 . The method of claim 1 , wherein assigning coded fluorescence patterns to a plurality of target analytes distinguishes at least 1,000 target analytes using no more than two populations of fluorophore labeled translator connector elements.