The invention relates to methods of multiplex detection of a plurality of target nucleic acids by contacting a sample with an acid reagent to remove bound nucleic acid detection systems, thereby allowing the same detection systems to be used again to detect different target nucleic acids and to provide for higher levels of multiplexing. The invention also relates to kits containing an acid reagent and optionally probes for detection of target nucleic acids.

The invention relates to methods of multiplex detection of a plurality of target nucleic acids by contacting a sample with an acid reagent to remove bound nucleic acid detection systems, thereby allowing the same detection systems to be used again to detect different target nucleic acids and to provide for higher levels of multiplexing. The invention also relates to kits containing an acid reagent and optionally probes for detection of target nucleic acids.

Provided herein is a method for mapping rolling circle amplification (RCA) products that contain unique identifier sequences. The method generally involves (a) producing a complex comprising population of grid oligonucleotide molecules and a population of RCA products that each have a unique RCA product identifier sequence, wherein the grid oligonucleotides are hybridized directly or indirectly via a splint to complementary sites in the RCA products; (b) extending the grid oligonucleotide molecules that are hybridized to two RCA products to add the complements of the unique RCA product identifier sequences from the two RCA products to the grid oligonucleotide molecules; (c) sequencing the extended grid oligonucleotides; (d) analyzing the sequences to identify which pairs of unique RCA product identifier sequence complements have been added onto the grid oligonucleotides; and (e) making one or more physical maps of the immobilized RCA products using the pairs of sequences identified in (d).

Provided herein is a method for mapping rolling circle amplification (RCA) products that contain unique identifier sequences. The method generally involves (a) producing a complex comprising population of grid oligonucleotide molecules and a population of RCA products that each have a unique RCA product identifier sequence, wherein the grid oligonucleotides are hybridized directly or indirectly via a splint to complementary sites in the RCA products; (b) extending the grid oligonucleotide molecules that are hybridized to two RCA products to add the complements of the unique RCA product identifier sequences from the two RCA products to the grid oligonucleotide molecules; (c) sequencing the extended grid oligonucleotides; (d) analyzing the sequences to identify which pairs of unique RCA product identifier sequence complements have been added onto the grid oligonucleotides; and (e) making one or more physical maps of the immobilized RCA products using the pairs of sequences identified in (d).

A method for sequencing a population of nucleic acids, which includes (a) binding the population of nucleic acids with a fractionally labeled mixture of nucleotides, thereby forming a fractionally labeled population of nucleic acids, wherein the mixture includes nucleotide cognates for a common base type in the templates, and wherein a fraction of the nucleotide cognates for the common base type in the mixture are exogenously labeled nucleotides that produce a signal that is not produced by other nucleotide cognates for the common base type in the mixture; (b) detecting the signal from the fractionally labeled population of nucleic acids; and (c) repeating (a) and (b) using a second mixture of the fractionally labeled nucleotides, wherein the fraction of the exogenously labeled nucleotides is higher in the second mixture.

Provided herein, among other things, is a method for analyzing a sample. In some embodiments, the method makes use of a plurality of binding agents that are each linked to a different oligonucleotide, as well as a corresponding plurality of peroxidase-linked oligonucleotides, wherein each of the peroxidase-linked oligonucleotides specifically hybridizes with only one of the binding agent-linked oligonucleotides. In some embodiments the method may comprise labeling the sample with the plurality of binding agents en masse, and then staining the sample by hybridizing a single peroxidase-linked oligonucleotide with the sample to produce complexes that comprise the peroxidase and then treating the sample with at least one tyramide-label conjugate. The peroxidase in the complexes activates the conjugate and causes covalent binding of the label to the sample near the complexes. Reagents and kits for performing the method are also provided.

Provided herein, among other things, is a method for analyzing a sample. In some embodiments, the method makes use of a plurality of binding agents that are each linked to a different oligonucleotide, as well as a corresponding plurality of peroxidase-linked oligonucleotides, wherein each of the peroxidase-linked oligonucleotides specifically hybridizes with only one of the binding agent-linked oligonucleotides. In some embodiments the method may comprise labeling the sample with the plurality of binding agents en masse, and then staining the sample by hybridizing a single peroxidase-linked oligonucleotide with the sample to produce complexes that comprise the peroxidase and then treating the sample with at least one tyramide-label conjugate. The peroxidase in the complexes activates the conjugate and causes covalent binding of the label to the sample near the complexes. Reagents and kits for performing the method are also provided.

Methods of detecting target polynucleotide sequences may include introducing one or more nucleic acid analytes to a first reaction mixture comprising a single-stranded probe oligonucleotide A.sub.0, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A.sub.0 to create a first intermediate product which is at least partially double-stranded, where the 3′ end of A.sub.0 forms a double-stranded complex with the analyte and where A.sub.0 is pyrophosphorylsed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A.sub.1. A.sub.1 may undergo ligation to form oligonucleotide A.sub.2. The methods may also include detecting a signal derived from the formed oligonucleotides, and inferring therefrom the presence or absence of the target polynucleotide sequence in the analyte.

Methods of detecting target polynucleotide sequences may include introducing one or more nucleic acid analytes to a first reaction mixture comprising a single-stranded probe oligonucleotide A.sub.0, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A.sub.0 to create a first intermediate product which is at least partially double-stranded, where the 3′ end of A.sub.0 forms a double-stranded complex with the analyte and where A.sub.0 is pyrophosphorylsed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A.sub.1. A.sub.1 may undergo ligation to form oligonucleotide A.sub.2. The methods may also include detecting a signal derived from the formed oligonucleotides, and inferring therefrom the presence or absence of the target polynucleotide sequence in the analyte.

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. Through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci. In some embodiments, nucleic acid probes include a signal moiety connected with a binding sequence via a cleavable linker.