Single-Molecule DNA Sequencing via Hybridization Blockage

The patent extracts the detection function from the nucleotide labels themselves and transfers it to the polymerase enzyme. The enzyme carries the fluorescent label and incorporates it during DNA synthesis, allowing detection without requiring labeled nucleotides. This separation of detection function from the nucleotide substrate resolves the contradiction between detection capability and cost.

The polymerase enzyme acts as an intermediary that carries the fluorescent label and transfers it to the growing DNA chain during incorporation. This intermediary approach allows the detection signal to be introduced through the enzyme rather than through expensive labeled nucleotides, reducing costs while maintaining detection precision.

The patent employs single-molecule sequencing where each DNA molecule is sequenced independently without requiring amplification. The detection system is sensitive enough to detect signals from individual molecules, eliminating the need for PCR or other amplification steps. This self-service approach where each molecule stands alone resolves the contradiction between material availability and time loss.

The patent replaces the biochemical amplification system (PCR) with a direct single-molecule detection system. Instead of mechanically amplifying DNA through enzymatic reactions, the system directly detects individual molecules using fluorescently labeled polymerases, eliminating the time-consuming amplification step while maintaining sufficient signal for detection.

Each DNA molecule is sequenced independently as a single unit without amplification. This independence prevents error propagation that occurs during PCR amplification, where a single error can be replicated across millions of copies. The self-service single-molecule approach maintains reliability by avoiding the error-prone amplification process entirely.

The patent extracts the sequencing capability from bulk amplified DNA and applies it to individual single molecules. By taking out the amplification step entirely and working directly with single molecules, the method eliminates the source of contamination and errors that arise during enzymatic amplification, thereby improving reliability.

The patent segments the sequencing process into independent single-molecule reactions that can be performed in parallel. Each DNA molecule is attached to an individual bead, creating thousands of independent reaction sites on a single bead. This segmentation allows simultaneous sequencing of multiple molecules without interference, dramatically increasing productivity while maintaining sequence determination accuracy.

The patent transitions from linear sequential sequencing to three-dimensional parallel processing by attaching multiple DNA molecules to the surface of microbeads. This dimensional change from one-dimensional linear processing to three-dimensional spatial parallelization enables thousands of sequences to be determined simultaneously, resolving the contradiction between precision and productivity.