METHODS AND COMPOSITIONS FOR DETERMINING LIGANDS ON MATRICES USING INDICES AND BARCODES Russian patent published in 2024 - IPC C12N15/10 C12Q1/6827 C12Q1/6869 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A method for detecting a plurality of target ligands in a matrix is described, including: (a) obtaining a substrate having a matrix of granules distributed on the surface of the specified substrate, wherein the specified matrix of granules contains the first and second subpopulation of granules, including: (i) obtaining the first and second subpopulations of granules, each granule containing: a capture probe, which capable of specifically binding to the target ligand, the first polynucleotide, which contains a barcode and a primer binding site at the 3' end relative to the specified barcode, while the specified barcode indicates a capture probe, and the second polynucleotide, containing an index and an index primer binding site at the 3' end relative to the index, each the index of the first subpopulation of granules differs from each index of the second subpopulation of granules; (ii) ensuring specific binding of the first target ligands to the specified capture probes of the specified first subpopulation of granules in the first reaction volume, and specific binding of the second target ligands to capture probes of the specified second subpopulation of granules in the second reaction volume, and (iii) distribution of the specified the first and second subpopulations of granules containing specifically bound first and second target ligands on the substrate, thereby obtaining a substrate having a matrix of granules; (b) detection in the specified matrix of specifically bound first and second target ligands of the specified first and second subpopulation of granules; (c) sequencing the barcodes of the specified first and second subpopulations of granules by hybridizing a plurality of primers with the primer binding sites of the barcodes of the specified first and second subpopulations of granules; and elongation of hybridized primers, thereby determining the position of the capture probes of the specified first and second subpopulations of granules in the specified matrix, (d) sequencing the indices of the specified first and second subpopulations of granules, thereby determining the positions of the specified first and second subpopulations of granules in the matrix; and (e) decoding the positions of granules in the specified matrix of granules, including the detected the target ligand in the matrix, thereby ensuring the identification of the target ligands of the specified first target ligands or the specified second target ligands in the matrix. A method for identifying target ligands in the matrix is also described, including: (a) obtaining the first and second subpopulations of granules, wherein each granule contains: (i) a capture probe that is capable of specifically binding to the target ligand, (ii) the first polynucleotide containing a barcode, wherein the specified barcode indicates a capture probe, and (iii) a second polynucleotide containing an index, wherein each index of the first subpopulation of granules differs from each index of the second subpopulation of granules; (b) ensuring specific binding of the first target ligands to the specified capture probes of the specified first subpopulation of granules in the first reaction volume, and specific binding of the second target ligands to capture probes of the specified second subpopulation of granules in the second reaction volume, and (c) distribution of the first and second subpopulations of granules containing specifically bound first and second target ligands on the substrate surface, thereby obtaining a matrix of granules; (d) detection of the specifically bound first and second target ligands of the specified first and second subpopulations in the specified matrix granules; (e) sequencing the barcodes of the pellet matrix; (f) sequencing the indices of the pellet matrix; and (g) decoding the positions of the granules in the specified pellet matrix, including the detected target ligand in the matrix, thereby ensuring the identification of target ligands in the pellet matrix.

EFFECT: invention expands the arsenal of methods for detecting specific nucleotide sequences present in a biological sample.

24 cl, 8 dwg, 4 ex