On-slide staining by primer extension
Inventors
Samusik, Nikolay • Nolan, Garry P. • Goltsev, Yury • McIlwain, David Robert
Assignees
Leland Stanford Junior University
Publication Number
US-12331344-B2
Publication Date
2025-06-17
Expiration Date
2034-12-04
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Abstract
A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.
Core Innovation
The invention provides a method for analyzing planar samples, such as tissue sections, using on-slide labeling and detection by primer extension. The method comprises labeling the planar sample with a capture agent that is linked to a nucleic acid containing an overhang sequence. The capture agent, such as an antibody, specifically binds to complementary sites within the planar sample, and the nucleic acid serves as a unique tag that can be extended, allowing for the detection of capture agent binding events.
After labeling, a labeled oligonucleotide that contains a fluorescent dye optical label is hybridized to the overhang sequence of the nucleic acid. The labeled oligonucleotide is then ligated to the nucleic acid using a ligase, such as T4 ligase. By using fluorescence microscopy, a signal generated by the labeled oligonucleotide can be read, allowing spatial visualization and mapping of the capture agent's binding pattern in the planar sample.
This approach addresses the limitations of existing single-cell antigen cytometry technologies, such as limited multiplexing capacity and challenges in analyzing archived or slide-based samples. The disclosed method enables flexible and multiplexed analysis of planar samples, including formalin-fixed, paraffin-embedded (FFPE) tissue, by combining nucleic acid labeling, selective extension, and fluorescence-based readout.
Claims Coverage
The patent includes a single independent claim detailing the main inventive features related to the on-slide labeling, oligonucleotide ligation, and fluorescence-based detection method.
Planar sample analysis by capture agent-linked nucleic acid and labeled oligonucleotide ligation
A method for analyzing a planar sample comprises: 1. Labeling the planar sample with a capture agent (comprising an antibody) linked to a nucleic acid that contains an overhang sequence. 2. Hybridizing a labeled oligonucleotide—containing a fluorescent dye optical label—to at least a portion of the overhang sequence. 3. Contacting the labeled sample with a ligase, resulting in the ligation of the labeled oligonucleotide to the nucleic acid. 4. Reading a signal generated by the labeled oligonucleotide through fluorescence microscopy. Key aspects include the usage of a capture agent linked to a nucleic acid with an overhang, the hybridization and ligation of a fluorescently labeled oligonucleotide, and detection by fluorescence microscopy.
The inventive features define a method that enables spatially resolved analysis of planar samples using capture agent-linked nucleic acids, targeted ligation of optically labeled oligonucleotides, and fluorescence readout, particularly emphasizing applicability to antibody-based detection in tissue sections.
Stated Advantages
Provides sensitive antigen detection and enables multiplexed analysis of planar samples, overcoming limitations of current cytometry approaches regarding multiplexing levels and analysis of archived or slide-based samples.
Allows detection of complementary sites, such as specific antigens, with high specificity and spatial resolution using fluorescence microscopy.
Applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections, expanding the method's use to clinical and archival specimens.
Documented Applications
Analysis of planar samples including tissue sections, sheets of cells, spun-down cells, blots of electrophoresis gels, Western blots, dot-blots, ELISAs, antibody microarrays, and nucleic acid microarrays.
Simultaneous detection and analysis of multiple biomarkers and cancer markers in formalin-fixed, paraffin-embedded (FFPE) tissue sections for diagnostic purposes.
Detection and spatial localization of DNA molecules and/or RNA molecules in situ, including single molecule RNA detection in cells.
Determination of the degree of dysplasia in cancer cells, identification and distinction of cancer cell types, and monitoring disease progression or treatment response.
Use in diagnostic, drug discovery, and research applications such as disease diagnosis, marker discovery, drug screening, determination of drug susceptibility, and basic cellular research.
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