The present invention provides a method for detecting a plurality of analytes in a sample, comprising performing a multiplex proximity-based detection assay. The assay utilises pairs of proximity probes with shared hybridisation sites (i.e. hybridisation sites which are shared between different proximity probe pairs). Also provided is a product comprising a plurality of proximity probe pairs with shared hybridisation sites, which may be used in the method disclosed herein.

A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.

A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.

The reaction-medicated amplification methods and applications include a new type of ligase. The general amplification and detection of the downstream with the ligase reaction includes 3 linking probes. It achieves the effect of eliminating nonspecific signal interference by respectively filling the detection tag sequence, upstream primer tag sequence, and downstream primer combination tag sequence into 3 different linking probes. Wherein the linking probe containing detection tag sequence forms a cystic structure and the specific hybridization sequences on both sides of the cystic structure form a “hybridization community” when being hybrid to the target sequences. Being hybrid closely at the adjacent positions to the target sequences, 3 linking probes finally form a complete probe chain containing 3 “tag” sequences with the effect of ligase. This technique achieves the goals of reducing reaction background, enhancing signal-noise-ration and avoiding false positive.

The reaction-medicated amplification methods and applications include a new type of ligase. The general amplification and detection of the downstream with the ligase reaction includes 3 linking probes. It achieves the effect of eliminating nonspecific signal interference by respectively filling the detection tag sequence, upstream primer tag sequence, and downstream primer combination tag sequence into 3 different linking probes. Wherein the linking probe containing detection tag sequence forms a cystic structure and the specific hybridization sequences on both sides of the cystic structure form a “hybridization community” when being hybrid to the target sequences. Being hybrid closely at the adjacent positions to the target sequences, 3 linking probes finally form a complete probe chain containing 3 “tag” sequences with the effect of ligase. This technique achieves the goals of reducing reaction background, enhancing signal-noise-ration and avoiding false positive.

Sensitive and specific detection of nucleic acids can be achieved using a chemical ligation-based template assisted rapid assay (TARA-L) with simple chemical reactions between probes and without the need for enzymes. Probes are designed to form a ligation product when they anneal to adjacent portions of a target nucleic acid. The ligation products can be detected, such as in immunochromatographic assays. The methods allow for the fast, efficient analysis of biological samples for the presence of nucleic acids and can be used, for example, in point of care settings.

Sensitive and specific detection of nucleic acids can be achieved using a chemical ligation-based template assisted rapid assay (TARA-L) with simple chemical reactions between probes and without the need for enzymes. Probes are designed to form a ligation product when they anneal to adjacent portions of a target nucleic acid. The ligation products can be detected, such as in immunochromatographic assays. The methods allow for the fast, efficient analysis of biological samples for the presence of nucleic acids and can be used, for example, in point of care settings.

Methods for visualizing modified nucleotides in a specific nucleic acid sequence or specific nucleic acid sequence interactions in single cells, wherein the methods comprise coupling an in situ hybridization (ISH) reaction with a proximity ligation as say (PLA) reaction.

Methods for visualizing modified nucleotides in a specific nucleic acid sequence or specific nucleic acid sequence interactions in single cells, wherein the methods comprise coupling an in situ hybridization (ISH) reaction with a proximity ligation as say (PLA) reaction.

The present invention provides assay systems and methods for detection of copy number variation at one or more loci and polymorphism detection at one or more loci in a mixed sample from an individual.