On-Slide Primer Extension Staining for Multiplexed Tissue Analysis

The patent introduces DNA oligonucleotides as intermediary molecules that bridge capture agents (antibodies) and detection systems. Each capture agent is conjugated to a unique DNA oligonucleotide sequence, which serves as a barcode that can be detected through PCR amplification and sequencing. This intermediary DNA layer enables high-throughput multiplexed analysis of hundreds to thousands of parameters simultaneously, far exceeding the 100-parameter limit of traditional fluorescence cytometry, while maintaining a relatively simple workflow using standard molecular biology techniques.

The patent creates a universal detection platform that can analyze multiple sample types including fresh cells, archived tissue sections, and slide-based samples. The method uses DNA oligonucleotide barcodes attached to capture agents that bind to antigens in situ within tissue sections, allowing the same workflow to process diverse sample formats. After antigen capture, the DNA barcodes are amplified by PCR and detected via sequencing, enabling high-throughput analysis of hundreds of samples in parallel while maintaining compatibility with archived formalin-fixed paraffin-embedded (FFPE) tissues that cannot be analyzed by traditional single-cell sequencing.

The patent performs preliminary action by attaching unique DNA oligonucleotide barcodes to capture agents (antibodies) before they contact the sample. This pre-conjugation step ensures that each capture agent carries its specific genetic identifier, enabling direct PCR amplification and sequencing detection without requiring complex in-situ synthesis or modification steps during the detection phase. The DNA barcodes are designed with specific sequences that allow selective primer binding and amplification, providing high sensitivity and specificity while keeping the detection workflow relatively simple using standard PCR and sequencing technologies.