The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

The present disclosure relates, in some embodiments, to isolated nucleic acids (also referred to as cap guides) and methods of use thereof for RNA mapping. The disclosure is based, in part, on guide RNAs that bind to a position that is at least 7 nucleotides downstream of the first nucleotide of an mRNA molecule.

The present disclosure relates, in some embodiments, to isolated nucleic acids (also referred to as cap guides) and methods of use thereof for RNA mapping. The disclosure is based, in part, on guide RNAs that bind to a position that is at least 7 nucleotides downstream of the first nucleotide of an mRNA molecule.

The present invention pertains to hybrid RNase H2 proteins that include fragments of amino acid sequences from Pyrococcus abyssi (P.a.), Thermococcus kodakarensis (T.kod), and Pyrococcus furiosus organisms, as well as methods of using the same to improve mismatch discrimination and activity in a high-fidelity DNA polymerase buffer.

The present invention pertains to hybrid RNase H2 proteins that include fragments of amino acid sequences from Pyrococcus abyssi (P.a.), Thermococcus kodakarensis (T.kod), and Pyrococcus furiosus organisms, as well as methods of using the same to improve mismatch discrimination and activity in a high-fidelity DNA polymerase buffer.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

The present invention relates to the field of genotyping samples containing short tandem repeat (STR) loci. More specifically, the present invention discloses a composition of matter containing an array of probes and a method to genotype these loci relying on the recognition of RNA:DNA base pairing followed by cleavage of the RNA containing strand. By measuring the temperature at which the chimeric DNA-RNA-DNA probe is cleaved, resulting in an increase of fluorescence of the probe, it can be assessed whether or not the probe and the sample share the same amount of repeats. An array of probes is utilised, covering all possible alleles of the investigated STR-locus. The probes and method of the present invention are well-suited to be used in a portable, less-expensive DNA analysis device and can be applied in other fields than forensics, like food fraud, diagnostics and many others.

The present invention relates to the field of genotyping samples containing short tandem repeat (STR) loci. More specifically, the present invention discloses a composition of matter containing an array of probes and a method to genotype these loci relying on the recognition of RNA:DNA base pairing followed by cleavage of the RNA containing strand. By measuring the temperature at which the chimeric DNA-RNA-DNA probe is cleaved, resulting in an increase of fluorescence of the probe, it can be assessed whether or not the probe and the sample share the same amount of repeats. An array of probes is utilised, covering all possible alleles of the investigated STR-locus. The probes and method of the present invention are well-suited to be used in a portable, less-expensive DNA analysis device and can be applied in other fields than forensics, like food fraud, diagnostics and many others.

The invention relates to a clustered regularly interspaced short palindromic repeats (CRISPR) based ribonucleic acid detection system. The invention further relates to a method for determining presence or absence of a target nucleic acid molecule in a sample with the aid of such system, and to a device comprising the ribonucleic acid detection system according to the invention.