The present disclosure provides methods and systems for sequencing nucleic acid molecules in a manner that enables higher sequencing accuracy. Methods and systems provided herein may enable sequences that may have low-accuracy reads, such as homopolymer sequences or other repeating sequences, to be determined at a higher accuracy and efficiency.

There is disclosed a kit for detecting a single strand target nucleotide sequence comprising: at least one first nucleic acid probe from 10 to 14 bases, to the 5′ end of which at least one fluorophore is bound; at least one second nucleic acid probe from 35 to 50 bases, comprising, from the 5′ to the 3′ end: a first segment having a nucleotide sequence complementary to the first nucleic acid probe, at least one quencher, and a second segment having a nucleotide sequence complementary to at least part of the target nucleotide sequence, wherein the following relation is met:
|ΔG hybr.target3−probe2|>|ΔG hybr.probe1−probe2|.

There is disclosed a kit for detecting a single strand target nucleotide sequence comprising: at least one first nucleic acid probe from 10 to 14 bases, to the 5′ end of which at least one fluorophore is bound; at least one second nucleic acid probe from 35 to 50 bases, comprising, from the 5′ to the 3′ end: a first segment having a nucleotide sequence complementary to the first nucleic acid probe, at least one quencher, and a second segment having a nucleotide sequence complementary to at least part of the target nucleotide sequence, wherein the following relation is met:
|ΔG hybr.target3−probe2|>|ΔG hybr.probe1−probe2|.

The present invention provides an oligonucleotide probe for single nucleotide polymorphism detection to be used for a target nucleic acid where a single nucleotide polymorphism is present, the oligonucleotide probe comprising a reporter region, an anchor region, and a linker region. The reporter region comprises: an oligonucleotide consisting of a sequence perfectly matching when a nucleotide of the single nucleotide polymorphism is a first nucleotide, and mismatching when the nucleotide of the single nucleotide polymorphism is a nucleotide other than the first nucleotide; and a fluorescent dye quenching when the reporter region hybridize to the target nucleic acid.

The present invention provides an oligonucleotide probe for single nucleotide polymorphism detection to be used for a target nucleic acid where a single nucleotide polymorphism is present, the oligonucleotide probe comprising a reporter region, an anchor region, and a linker region. The reporter region comprises: an oligonucleotide consisting of a sequence perfectly matching when a nucleotide of the single nucleotide polymorphism is a first nucleotide, and mismatching when the nucleotide of the single nucleotide polymorphism is a nucleotide other than the first nucleotide; and a fluorescent dye quenching when the reporter region hybridize to the target nucleic acid.

Provided herein are reagents and kits for detection of multiple target sequences in a single-tube, single-color assay, and methods of use thereof. In particular, multiplex assays are provided for the detection of Mycobacterium tuberculosis complex target sequences (e.g., katG, rpoB, inhA promotor, pncA, etc.).

Provided herein are reagents and kits for detection of multiple target sequences in a single-tube, single-color assay, and methods of use thereof. In particular, multiplex assays are provided for the detection of Mycobacterium tuberculosis complex target sequences (e.g., katG, rpoB, inhA promotor, pncA, etc.).

Provided is a method for detecting target substances. The method includes a) introducing a sample containing target substances onto a substrate, b) allowing detection probes conjugated with docking strands to specifically bind to the target substances, c) introducing one or more separate strands capable of complementary binding to the docking strands into the docking strands, either the docking strands or the separate strands, or both, are labeled with at least one donor fluorescent substance and at least one acceptor fluorescent substance, and d) measuring fluorescence signals generated by the FRET between the donor fluorescent substance and the acceptor fluorescent substance to identify the target substances.

Provided is a method for detecting target substances. The method includes a) introducing a sample containing target substances onto a substrate, b) allowing detection probes conjugated with docking strands to specifically bind to the target substances, c) introducing one or more separate strands capable of complementary binding to the docking strands into the docking strands, either the docking strands or the separate strands, or both, are labeled with at least one donor fluorescent substance and at least one acceptor fluorescent substance, and d) measuring fluorescence signals generated by the FRET between the donor fluorescent substance and the acceptor fluorescent substance to identify the target substances.

Fluorescence resonance energy transfer (FRET)-based nucleic acid sensors and methods of their use for detecting nucleic acids are provided. The sensors are highly sensitive and detect nucleic acids at the femtomolar (fM) level, without the need for labeling and amplification.