Detection of Nucleic Acids and Proteins
Methods of detecting various types of nucleic acids, including methods of detecting two or more nucleic acids in multiplex branched-chain DNA assays, are provided. Detection assays may be conducted at least in vitro, in cellulo, and in situ. Nucleic acids which are optionally captured on a solid support are detected, for example, through cooperative hybridization events that result in specific association of a label probe system with the nucleic acids. Various label probe system embodiments are provided. Embodiments are directed to concurrent detection of one or more nucleic acids and one or more proteins. Embodiments also are directed to determining the methylation state of a target sequence. Other embodiments are directed to detection of one or more proteins using DNA barcodes. Compositions, kits, and systems related to the methods are also described.
1 . A method of detecting a nucleic acid and protein, which comprises:
providing a sample comprising or suspected of comprising a target nucleic acid and a target protein;
incubating at least two label extender probes each comprising a different L-1 sequence, an antibody specific for the target protein, and at least two label probe systems with the sample comprising or suspected of comprising the target nucleic acid and the target protein, wherein the antibody comprises a pre-amplifier probe, and wherein the at least two label probe systems each comprise a detectably different label; and
detecting the detectably different labels in the sample.
2 . The method according to claim 1 , wherein the at least one L-1 sequence comprises one or more locked nucleic acids.
3 . The method according to claim 2 , wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).
4 . The method according to claim 1 , wherein the target is selected from one or more of the group consisting essentially of: double-stranded DNA, miRNA, siRNA, mRNA, and single-stranded DNA.
5 . The method according to claim 1 , wherein the method is performed in situ.
6 . The method according to claim 1 , wherein the sample is cells obtained from a cell culture.
7 . The method according to claim 1 , wherein the sample comprises or is suspected of comprising at least two different target nucleic acids or at least two different target proteins.
8 . The method according to claim 1 , wherein the label extenders are designed in the cruciform orientation.
9 . The method according to claim 1 , wherein the target nucleic acid encodes the target protein.
10 . The method according to claim 1 , wherein the physical location and quantity of the target nucleic acid and the target protein within a cell or tissue is detected.
11 . A method of detecting a protein, which comprises:
providing a sample comprising or suspected of comprising a target protein;
incubating an antibody with the sample, wherein the antibody comprises at least one pre-amplifier probe sequence;
incubating at least one label probe system with the sample; and
detecting whether the label probe system is associated with the sample.
12 . The method according to claim 11 , wherein the at least one component of the label probe system comprises one or more locked nucleic acids.
13 . The method according to claim 12 , wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).
14 . The method according to claim 11 , wherein the label probe system comprises one or more label extenders which are designed in the cruciform orientation.
15 . A method of detecting a protein, which comprises:
providing a sample comprising or suspected of comprising a target protein;
incubating an antibody with the sample, wherein the antibody comprises at least one barcode probe sequence;
isolating the antibodies which bind to the sample; and
identifying the at least one barcode probe sequence which specifically bound to the sample, thereby detecting the protein in the sample.
16 . The method according to claim 15 , wherein isolating the antibodies which bind to the system further comprises:
washing the sample;
eluting the antibodies specifically bound to the sample;
cleaving the at least one barcode sequence; and
sequencing the barcode sequence.
17 . The method according to claim 15 , wherein identifying the at least one barcode probe sequence comprises hybridizing the at least one barcode probe sequence to a microarray, thereby identifying the at least one barcode sequence.
18 . A method of determining the methylation state of a nucleic acid sequence, which comprises:
providing a sample comprising or suspected of comprising a target nucleic acid sequence;
incubating at least two pairs of label extender probes each comprising a different L-1 sequence, at least one pre-amplifier comprising a sequence which is complementary to the target sequence in a region where the methylation status is unknown, and at least three label probe systems with the sample, wherein the at least three label probe systems each comprise a detectably different label; and
detecting the detectably different labels in the sample.
19 . The method according to claim 18 , wherein at least one L-1 sequence comprises one or more locked nucleic acids.
20 . The method according to claim 19 , wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).
21 . The method according to claim 18 , wherein the method is performed in situ.
22 . The method according to claim 18 , wherein the sample is cells obtained from a cell culture.
23 . The method according to claim 18 , wherein the sample comprises or is suspected of comprising at least two different target nucleic acids.
24 . The method according to claim 18 , wherein one or more of the label extenders are designed in the cruciform orientation.