Compositions and Methods for Analyte Detection
Methods of analyzing nucleic acids of a cell are provided.
1 .- 30 . (canceled)
31 . A method, comprising:
(a) contacting a cell or tissue sample comprising an analyte with a detection reagent to bind said detection reagent to said analyte in said cell or tissue sample, wherein said detection reagent comprises (i) a probe that binds to said analyte and (ii) one or more predetermined subsequences; and
(b) detecting said one or more predetermined subsequences in said cell or tissue sample in a temporally sequential manner to obtain a plurality of signal signatures associated with said analyte.
32 . The method of claim 31 , wherein said one or more predetermined subsequences is a plurality of predetermined subsequences.
33 . The method of claim 32 , wherein detecting said plurality of predetermined subsequences in said cell or tissue sample in said temporally sequential manner comprises:
(i) binding a first decoder probe to a first predetermined sequence of said plurality of predetermined subsequences, wherein said first decoder probe comprises a first detectable label;
(ii) detecting said first detectable label to obtain a first signal signature of said plurality of signal signatures;
(iii) removing said first signal signature from said cell or tissue sample;
(iv) binding a second decoder probe to a second predetermined sequence of said plurality of predetermined subsequences, wherein said second decoder probe comprises a second detectable label; and
(v) detecting said second detectable label to obtain a second signal signature of said plurality of signal signatures.
34 . The method of claim 33 , wherein said first detectable label is a first optical label, wherein said second detectable label is a second optical label, and wherein said plurality of signal signatures comprises at least one signal signature characterized by an absence of an optical signal.
35 . The method of claim 33 , wherein: (i) said first detectable label is a first fluorescent label and said first signal signature is a first fluorescent color associated with said first fluorescent label; (ii) said second detectable label is a second fluorescent label and said second signal signature is a second fluorescent color associated with said second fluorescent label; and (iii) said plurality of signal signatures comprises at least one signal signature associated with no fluorescent color.
36 . The method of claim 35 , wherein said first fluorescent color and said second fluorescent color are the same fluorescent color.
37 . The method of claim 35 , wherein said first fluorescent color and said second fluorescent color are different fluorescent colors.
38 . The method of claim 31 , wherein said plurality of signal signatures comprises at least five signal signatures.
39 . The method of claim 38 , wherein said plurality of signal signatures comprises at least ten signal signatures.
40 . The method of claim 31 , wherein said cell or tissue sample is a plurality of fixed cells immobilized on a solid support.
41 . The method of claim 31 , wherein said cell or tissue sample is a fixed tissue section immobilized on a solid support.
42 . The method of claim 35 , wherein said first fluorescent label and said second fluorescent label are directly attached to said first decoder probe and said second decoder probe, respectively, via a covalent bond.
43 . The method of claim 35 , wherein said first fluorescent label and said second fluorescent label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via a non-covalent bond.
44 . The method of claim 35 , wherein said first fluorescent label and said second fluorescent label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via an intermediary molecule.
45 . The method of claim 35 , wherein said first fluorescent label and said second fluorescent label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via a binding molecule that hybridizes to said first decoder probe or said second decoder probe.
46 . The method of claim 35 , wherein removing said first signal signature from said cell or tissue sample comprises photo-bleaching, quenching, chemically degrading, bleaching, or oxidizing said first fluorescent label.
47 . The method of claim 33 , wherein removing said first signal signature from said cell or tissue sample comprises removing said first decoder probe from said cell or tissue sample by washing, heating, varying a salt concentration, using a detergent, using a denaturant, or any combination thereof.
48 . A method, comprising:
(a) providing a cell or tissue sample on a solid support, wherein said cell or tissue sample comprises a ribonucleic acid (RNA) analyte at a spatial location of said cell or tissue sample;
(b) contacting said cell or tissue sample with a detection reagent to bind said detection reagent to said RNA analyte at said spatial location, wherein said detection reagent is a nucleic acid molecule comprising: (i) a probe sequence that hybridizes to said RNA analyte and (ii) a plurality of predetermined subsequences; and
(c) detecting said plurality of predetermined sequences in said cell or tissue sample in a temporally sequential manner to obtain a plurality of signal signatures at said spatial location, wherein said detecting comprises:
(i) contacting said cell or tissue sample with a first decoder probe to bind said first decoder probe to a first predetermined subsequence of said plurality of predetermined subsequences, wherein said first decoder probe comprises a first detectable label;
(ii) detecting said first detectable label to obtain a first signal signature of said plurality of signal signatures at said spatial location;
(iii) removing said first signal signature from said cell or tissue sample;
(iv) contacting said cell or tissue sample with a second decoder probe to bind said second decoder probe to a second predetermined subsequence of said plurality of predetermined subsequences, wherein said second decoder probe comprises a second detectable label; and
(v) detecting said second detectable label to obtain a second signal signature of said plurality of signal signatures at said spatial location.
49 . The method of claim 48 , wherein said first detectable label is a first optical label, wherein said second detectable label is a second optical label, and wherein said plurality of signal signatures comprises at least one signal signature characterized by an absence of an optical signal at said spatial location.
50 . The method of claim 48 , wherein: (i) said first detectable label is a first fluorescent label and said first signal signature is a first fluorescent color associated with said first fluorescent label; (ii) said second detectable label is a second fluorescent label and said second signal signature is a second fluorescent color associated with said second fluorescent label; and (iii) said plurality of signal signatures comprises at least one signal signature at said spatial location associated with no fluorescent color.
51 . The method of claim 50 , wherein said first fluorescent color and said second fluorescent color are the same fluorescent color.
52 . The method of claim 50 , wherein said first fluorescent color and said second fluorescent color are different fluorescent colors.
53 . The method of claim 50 , wherein said first fluorescent label and said second fluorescent label are directly attached to said first decoder probe and said second decoder probe, respectively, via a covalent bond.
54 . The method of claim 50 , wherein said first fluorescent label and said second fluorescent label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via a non-covalent bond.
55 . The method of claim 50 , wherein said first fluorescent label and said second fluorescent label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via an intermediary molecule.
56 . The method of claim 50 , wherein said first fluorescent label and said second fluorescent label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via a binding molecule that hybridizes to said first decoder probe or said second decoder probe.
57 . The method of claim 48 , wherein said plurality of signal signatures comprises at least five signal signatures.
58 . The method of claim 57 , wherein said plurality of signal signatures comprises at least ten signal signatures.
59 . The method of claim 50 , wherein removing said first signal signature comprises photo-bleaching, quenching, chemically degrading, bleaching, or oxidizing said first fluorescent label.
60 . The method of claim 48 , wherein removing said first signal signature comprises removing said first decoder probe from said cell or tissue sample by washing, heating, varying a salt concentration, using a detergent, using a denaturant, or any combination thereof.