Oligonucleotide Probe Synthesis via Primer Extension

The patent applies preliminary action by pre-synthesizing and pooling oligonucleotide primers with variable sequences (V1 and V2) before the actual probe synthesis. These pre-prepared primers are stored as ready-to-use pools, eliminating the need for time-consuming de novo synthesis during the probe production process. The primer pools are designed in advance with known sequences that hybridize to specific genomic regions, enabling rapid probe generation when needed.

The probe synthesis process is segmented into distinct modular components: (1) pre-synthesized oligonucleotide primers with variable sequences, (2) a common complementary sequence (B/B') that serves as a universal binding site, and (3) the target genomic DNA. This segmentation allows each component to be optimized and prepared independently, then combined efficiently through hybridization and extension reactions, dramatically improving overall productivity.

The patent implements universality through the common sequence B/B' that serves multiple functions: it provides a universal hybridization site for all variable primers, acts as a common binding platform for the template strand, and enables all probe molecules to be synthesized through the same standardized extension process. This universal component allows the system to generate diverse probes targeting different genomic regions using a single unified synthesis protocol, thereby increasing adaptability without proportionally increasing complexity.

The methodology uses copying by employing identical common sequences (B and B') that can be replicated and paired with different variable sequences. The common sequence acts as a reusable template or scaffold that is copied alongside different V1-V2 primer combinations, enabling systematic generation of multiple probe variants from a single set of reagents. This copying approach simplifies the synthesis process by reducing the number of unique components that must be managed.