The present invention relates to the detection of a target nucleic acid sequence by a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay. The present invention adopts the occurrence of the inhibition of the hybridization between the HO with the CTO by the formation of the target-dependent extended duplex. Therefore, the present invention may detect target sequences even when the HO is not cleaved. In this regard, the design of the 5′-tagging portion of PTO, CTO and HO sequences may be readily performed and the conditions for reactions may be also easily established. In addition, the detection of the hybrid between the CTO and the HO may be performed in a different vessel from that for the extension of the CTO.

Detection method of a base mutation in a target base sequence of a nucleic acid sample, includes: performing a PCR reaction with the nucleic acid sample as a template, using a primer set capable of amplifying, by PCR, an amplification target region including the target base sequence; a blocker nucleic acid fragment having a base sequence complementary to the target base sequence and including a residue which is synthetic nucleic acid; and a probe that hybridizes to a region, in the target region, closer to a 5′ end of the target region than the target base sequence in the same chain as the target base sequence, and that has a fluorescent substance on one of a 5′ end and a 3′ end of the probe and a quenching substance on the other; and measuring an amplification amount of the template in the PCR reaction by detecting fluorescence from the probe.

Detection method of a base mutation in a target base sequence of a nucleic acid sample, includes: performing a PCR reaction with the nucleic acid sample as a template, using a primer set capable of amplifying, by PCR, an amplification target region including the target base sequence; a blocker nucleic acid fragment having a base sequence complementary to the target base sequence and including a residue which is synthetic nucleic acid; and a probe that hybridizes to a region, in the target region, closer to a 5′ end of the target region than the target base sequence in the same chain as the target base sequence, and that has a fluorescent substance on one of a 5′ end and a 3′ end of the probe and a quenching substance on the other; and measuring an amplification amount of the template in the PCR reaction by detecting fluorescence from the probe.

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.

Embodiments disclosed herein relate to methods and systems for analysis of melting temperatures, and particularly to analysis of duplex nucleic acids.

Embodiments disclosed herein relate to methods and systems for analysis of melting temperatures, and particularly to analysis of duplex nucleic acids.

Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.

Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.

Method of analysis for alleles of a target. In the method, a plurality of fluid volumes may be formed. Each fluid volume may contain a first primer pair to amplify a first allele of a target, a second primer pair to amplify a second allele of the target, a first reporter including a first photoluminophore and providing a primer of the first primer pair, and a second reporter including a second photoluminophore and providing a primer of the second primer pair. Each of the first and second reporters may include an oligomer having a quencher, and the oligomer may be configured to base-pair with the primer of the first primer pair and the primer of the second primer pair. The first and second alleles may be amplified using the first and second primer pairs. Photoluminescence may be detected. A level of each allele may be determined.