Detection of Nucleic Acids
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. Compositions, kits, and systems related to the methods are also described.
1 . A method of detecting a target nucleic acid sequence, which comprises:
providing a sample comprising or suspected of comprising a target nucleic acid sequence;
incubating at least two label extender probes each comprising a different L-1 sequence, and a label probe system with the sample comprising or suspected of comprising the target nucleic acid sequence; and
detecting whether the label probe system is associated with the sample.
2 . The method according to claim 1 , wherein the sample is purified chromosomes and the target is double stranded DNA.
3 . The method according to claim 1 , wherein the sample comprises or is suspected of comprising a target nucleic acid comprising at least two single nucleotide polymorphisms.
4 . The method according to claim 1 , wherein the label extender probes comprise an allele-specific label probe and a non-allele-specific probe and wherein the non-allele-specific probe is designed to hybridize less stringently to the complimentary strand of the target sequence.
5 . The method according to claim 1 , wherein the at least one L-1 sequence comprises one or more locked nucleic acids.
6 . The method according to claim 5 , wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).
7 . The method according to claim 1 , wherein the target nucleic acid sequence comprises one or more single nucleotide polymorphisms.
8 . The method according to claim 1 , wherein incubating at least two label extender probes each comprising a different L-1 sequence comprises incubating at least eight label extender probes each comprising a different L-1 sequence, wherein the at least eight label extender probes comprise two sets of four complimentary label extender probes designed to bind double stranded DNA target sequences, and wherein one set of four complimentary label extender probes binds downstream or upstream of the other set of four complimentary label extender probes.
9 . The method according to claim 8 , wherein each set of four complimentary label extender probes associates with a different set of label probes and each different set of label probes comprise a detectably distinguishable label.
10 . A method of detecting a pri-microRNA, which comprises:
providing a sample comprising or suspected of comprising a pri-microRNA;
incubating at least two sets of two label extender probes each comprising a different L-1 sequence, and a label probe system with the sample comprising or suspected of comprising the pri-microRNA, wherein at least one set of L-1 sequences is complementary to a pri-microRNA sequence comprising both stem-loop structure mature sequence and non-stem-loop structure pri-microRNA sequence;
detecting whether the label probe system is associated with the sample.
11 . The method according to claim 10 , wherein the sample is a tissue.
12 . The method according to claim 10 , wherein the sample is cells from a cell culture.
13 . The method according to claim 10 , wherein the cells are human cells.
14 . The method according to claim 10 , wherein the at least one L-1 sequence comprises one or more locked nucleic acids.
15 . The method according to claim 14 , wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).
16 . The method according to claim 10 , wherein the sample comprises both mature miRNA and pri-miRNA and wherein two differently labeled label probe systems are present, thereby detecting whether the sample comprises mature miRNA, immature miRNA or both.
17 . The method according to claim 10 , wherein at least two different pri-miRNA sequences are in the sample.
18 . The method according to claim 10 , wherein the label extenders are designed in the cruciform orientation.