The present disclosure describes a CRISPR nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The CRISPR cascade assays utilize signal amplification mechanisms comprising various components including CRISPR nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

The present disclosure describes a CRISPR nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The CRISPR cascade assays utilize signal amplification mechanisms comprising various components including CRISPR nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

Methods of determining methylation of DNA are provided. In one illustrative, but non-limiting embodiment the method comprises i) contacting a biological sample comprising a nucleic acid to a first matrix material comprising a first column or filter where said matrix material binds and/or filters nucleic acids in said sample and thereby purifies the DNA; ii) eluting the bound DNA from the first matrix material and denaturing the DNA to produce eluted denatured DNA; iii) heating the eluted DNA in the presence of bisulfite ions to produce a deaminated nucleic acid; iv) contacting said deaminated nucleic acid to a second matrix material comprising a second column to bind said deaminated nucleic acid to said second matrix material; v) desulphonating the bound deaminated nucleic acid and/or simultaneously eluting and desulphonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a bisulfite converted nucleic acid; vi) eluting said bisulfite converted nucleic acid from said second matrix material; and vii) performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on said bisulfite-converted nucleic acid to determine the methylation of said nucleic acid, wherein at least steps iv) through vi) are performed in a single reaction cartridge.

The present disclosure provides, in some aspects, nucleic acid-based molecular tools that enable the recording of molecular structure and soluble signals as well as the programmed assembly of molecular structures.

The present disclosure provides, in some aspects, nucleic acid-based molecular tools that enable the recording of molecular structure and soluble signals as well as the programmed assembly of molecular structures.

A method of detecting a ligase expressing micro-organism in a sample comprises steps of treating the sample under conditions that inhibit the activity of ATP-dependent ligase from mammalian cells but which do not inhibit the activity of the microbial ligases, contacting the sample or a portion of the sample with a nucleic acid molecule which acts as a substrate for ligase activity in the sample, incubating the thus contacted sample under conditions suitable for ligase activity; and specifically determining the presence and/or the amount of a ligated nucleic acid molecule resulting from the action of the ligase on the substrate nucleic acid molecule to indicate the presence of the ligase expressing micro-organism. The micro-organism may be a fungus or a bacterium or both. High pH conditions may be employed to inactivate mammalian ligases. Related kits are described.

This document provides methods and materials for detecting target nucleic acid. For example, methods and materials for detecting the presence or absence of target nucleic acid, methods and materials for detecting the amount of target nucleic acid present within a sample, kits for detecting the presence or absence of target nucleic acid, kits for detecting the amount of target nucleic acid present within a sample, and methods for making such kits are provided.

This document provides methods and materials for detecting target nucleic acid. For example, methods and materials for detecting the presence or absence of target nucleic acid, methods and materials for detecting the amount of target nucleic acid present within a sample, kits for detecting the presence or absence of target nucleic acid, kits for detecting the amount of target nucleic acid present within a sample, and methods for making such kits are provided.

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.

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.