Signal Multiplexing and Signal Amplification
Disclosed are methods, compositions and kits for amplifying signals for detecting the presence, quantity and/or sequence of nucleic acids and proteins, as well as methods, compositions and kits for increasing the number of such targets simultaneously detectable in a sample. Detection may be, for instance, in vivo, in cellulo or in situ. Amplification of signal is achieved by way of hybridization of nucleic acid label probe systems and structures. Increase in target multiplex capacity is achieved by way of varying the type of labels utilized in the nucleic acid label probe system.
1 . A method of amplifying a nucleic acid detection signal, which comprises:
hybridizing one or more label extender probes to a target nucleic acid;
hybridizing a pre-amplifier to the one or more label extender probes;
hybridizing one or more amplifiers to the pre-amplifier;
hybridizing one or more label spoke probes to the one or more amplifiers; and
hybridizing one or more label probes to the one or more label spoke probes.
2 . The method according to claim 1 , wherein the one or more label probes comprise one or more different labels.
3 . The method according to claim 1 , wherein the one or more label extender probes comprise one or more nucleic acid analogs.
4 . The method according to claim 3 , wherein the one or more nucleic acid analogs is cEt.
5 . The method according to claim 1 , which further comprises:
hybridizing one or more capture extender probes with the target nucleic acid; and
hybridizing the one or more capture extenders to one or more capture probes, wherein the capture probes are covalently attached to a substrate.
6 . The method according to claim 5 , wherein the substrate comprises one or more substrates of varying size, shape and color.
7 . The method according to claim 6 , wherein each one of the one or more substrates of varying size, shape and color is each associated with a different target nucleic acid.
8 . The method according to claim 1 , wherein the method is conducted in situ or in vitro.
9 . The method according to claim 1 , which further comprises:
providing a target protein;
hybridizing to the target protein a molecule possessing affinity for the target protein, wherein the molecule comprises covalently conjugated thereto a pre-amplifier probe;
hybridizing one or more amplifiers to the pre-amplifier;
hybridizing one or more label spoke probes to the one or more amplifiers; and
hybridizing one or more label probes to the one or more label spoke probes.
10 . A method of amplifying a protein detection signal, which comprises:
hybridizing a molecule possessing affinity for the target protein, wherein the molecule comprises covalently conjugated thereto a pre-amplifier probe;
hybridizing one or more amplifiers to the pre-amplifier;
hybridizing one or more label spoke probes to the one or more amplifiers; and
hybridizing one or more label probes to the one or more label spoke probes.
11 . The method according to claim 10 , wherein the one or more label probes comprise one or more different labels.
12 . The method according to claim 10 , wherein the target protein is immobilized on a substrate.
13 . The method according to claim 12 , wherein the substrate comprises one or more substrates of varying size, shape and color.
14 . The method according to claim 13 , wherein each one of the one or more substrates of varying size, shape and color is each associated with a different target nucleic acid.
15 . The method according to claim 10 , wherein the method is conducted in situ or in vitro.