This invention relates to methods for analyzing nucleic acids. The disclosure provides methods of a primer-dependent amplification and detection method that is capable of amplifying and detecting in a sample as few as ten copies of at least one rare intended target sequence in the presence of abundant closely related unintended target sequences. Also provided are reaction compositions and kits for performing the methods.

Provided herein are devices, methods, and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein.

Provided herein are devices, methods, and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein.

Methods of quantifying a species of RNA within a sample comprising, receiving a sample comprising RNA and substantially devoid of DNA, contacting the sample with a first primer to produce a first cDNA strand, treating the sample with RNAse, contacting the sample with a second primer to produce an amplification product, treating the sample with a proteinase, passing the amplification product through a nanopore and identifying the amplification product derived from the RNA as it passes through nanopore. Methods of diagnosing a disease in a subject are also provided.

Methods of quantifying a species of RNA within a sample comprising, receiving a sample comprising RNA and substantially devoid of DNA, contacting the sample with a first primer to produce a first cDNA strand, treating the sample with RNAse, contacting the sample with a second primer to produce an amplification product, treating the sample with a proteinase, passing the amplification product through a nanopore and identifying the amplification product derived from the RNA as it passes through nanopore. Methods of diagnosing a disease in a subject are also provided.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

The present disclosure relates to a method of target enrichment and amplification of selective target regions of genomes or nucleic acid samples and further analysis by systems such as NGS. The disclosure provides methods and kits that can be used in numerous downstream procedures including DNA sequencing. The disclosed methods can be utilized to sequence the target nucleic acid sequences such as to detect the presence of genetic variations for biological assays, assessment of disease, to count copies of target regions, and to allow such target enrichment prior to sequencing.