The invention relates to methods of detecting mutations associated with the Ras homologue gene family member (RHOA) gene, and diagnosing conditions associated with these mutations, using Competitive allele-specific TaqMan polymerase chain reaction (cast-PCR). The invention also extends to the products used to detect mutations, and their use in diagnosis.

The invention relates to methods of detecting mutations associated with the Ras homologue gene family member (RHOA) gene, and diagnosing conditions associated with these mutations, using Competitive allele-specific TaqMan polymerase chain reaction (cast-PCR). The invention also extends to the products used to detect mutations, and their use in diagnosis.

The present disclosure generally relates, in certain aspects, to droplet-based microfluidic devices and methods. In certain aspects, target nucleic acids contained within droplets are amplified within droplets in a first step, where multiple primers may be present. However, multiple primers may cause multiple target nucleic acids to be amplified within the droplets, which can make it difficult to identify which nucleic acids were amplified. In a second step, the amplified nucleic acids may be determined. For example, the droplets may be broken and the amplified nucleic acids can be pooled together and sequenced. As an example, new droplets may be formed containing the amplified nucleic acids, and those nucleic acids within the droplets amplified by exposure to certain primers.

The present disclosure generally relates, in certain aspects, to droplet-based microfluidic devices and methods. In certain aspects, target nucleic acids contained within droplets are amplified within droplets in a first step, where multiple primers may be present. However, multiple primers may cause multiple target nucleic acids to be amplified within the droplets, which can make it difficult to identify which nucleic acids were amplified. In a second step, the amplified nucleic acids may be determined. For example, the droplets may be broken and the amplified nucleic acids can be pooled together and sequenced. As an example, new droplets may be formed containing the amplified nucleic acids, and those nucleic acids within the droplets amplified by exposure to certain primers.

The present disclosure relates to compositions, methods, and kits for generating capture probes on a substrate for identifying the location of analytes in a biological sample. In particular, disclosed is a method of generating a spatial array comprising: (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein an acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate; (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle present in a donor oligonucleotide of a plurality of donor oligonucleotides; and (c) ligating the donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.

The present disclosure relates to compositions, methods, and kits for generating capture probes on a substrate for identifying the location of analytes in a biological sample. In particular, disclosed is a method of generating a spatial array comprising: (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein an acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate; (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle present in a donor oligonucleotide of a plurality of donor oligonucleotides; and (c) ligating the donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′db-adapter to the 3′-end of target RNAs, wherein the 3′-db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′db-adapter to the 3′-end of target RNAs, wherein the 3′-db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

The invention discloses a composition and kit for early detection of high-grade cervical lesions and cervical cancer, wherein the composition for early detection of high-grade cervical lesions and cervical cancer includes methylation primers, a probe corresponding to methylated sites and methylation blocking primers for FAM19A4 gene; methylation primers, a probe corresponding to methylated sites and methylation blocking primers for JAM3 gene; methylation primers, a probe corresponding to methylated sites and methylation blocking primers for PAX1 gene; and 1 pair of primers and a probe corresponding to methylated sites for internal reference gene GAPDH. The methylated sites in FAM19A4, JAM3 and PAX1 genes are accurately detected using multiple multi-channel fluorescence and blocking techniques through accurate recognition between specific primers and probes and methylated sequences, full release of methylated templates under the action of multiple blocking primers and optimized special methylation DNA polymerase.

The invention discloses a composition and kit for early detection of high-grade cervical lesions and cervical cancer, wherein the composition for early detection of high-grade cervical lesions and cervical cancer includes methylation primers, a probe corresponding to methylated sites and methylation blocking primers for FAM19A4 gene; methylation primers, a probe corresponding to methylated sites and methylation blocking primers for JAM3 gene; methylation primers, a probe corresponding to methylated sites and methylation blocking primers for PAX1 gene; and 1 pair of primers and a probe corresponding to methylated sites for internal reference gene GAPDH. The methylated sites in FAM19A4, JAM3 and PAX1 genes are accurately detected using multiple multi-channel fluorescence and blocking techniques through accurate recognition between specific primers and probes and methylated sequences, full release of methylated templates under the action of multiple blocking primers and optimized special methylation DNA polymerase.