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METHOD FOR NUCLEIC ACID DETECTION BY OLIGO HYBRIDIZATION AND PCR-BASED AMPLIFICATION
METHOD FOR NUCLEIC ACID DETECTION BY OLIGO HYBRIDIZATION AND PCR-BASED AMPLIFICATION
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机译:寡核苷酸杂交和PCR扩增检测核酸的方法
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摘要
The present invention relates to the field of nucleic acid sequencing at the single cell level, e.g., single-cell RNA sequencing (scRNA-seq). In particular, the invention provides a method of detecting nucleic acid in a fixated or non-fixated nucleic acid-containing compartment such as a eukaryotic cell or nucleus thereof, by hybridizing a plurality of single-stranded (ss)DNA oligonucleotide probes to complementary nucleic acid molecules within said compartment; removing ssDNA oligonucleotide probes from the compartment that have not specifically hybridized to nucleic acid; and identifying the ssDNA oligonucleotide probes specifically hybridized to nucleic acid molecules within said compartment by sequencing or amplification, thereby determining the corresponding nucleic acids present in said compartment. The method does not require a step of sequential ssDNA probe hybridization to the same target nucleic acid as a means for increased specificity or sensitivity, and preferably further does not require steps of RNA isolation and cDNA generation. The method of the invention has the potential to detect substantially every known and/or unknown nucleic acid species, in particular RNA, e.g., protein-encoding mRNAs as well as non-coding RNAs. The method further enables spatial mapping of detected nucleic acids, wherein the compartment is sectioned or dissociated into a single cell suspension prior to probe hybridization to obtain a collection of fractions and thus nucleic acid molecules are separated from each other depending on their localization or which cell type they belonged to. Spatial mapping of detected nucleic acids may be combined with the detection of at least one DNA locus, at least one protein, or with the analysis of chromatin condensation. The method of the invention is designated oligo-seq.
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