Sequence-specific oligonucleotide (SSO) methods use synthetic probes manufactured to bind to complementary strands of target exon DNA from the cells being assessed. These probes are combined with primers, buffers, and Taq polymerase for PCR amplification.
The general procedure is as follows:
- DNA is extracted and quantified
- DNA is mixed with the cocktail of primers, buffer, and Taq polymerase for PCR/thermocycling.
- PCR - In high temperatures, the double-stranded DNA separates and primers bind to their complementary target. The Taq polymerase synthesizes DNA from there in cooler temperatures; repeat, repeat, repeat.
- After PCR, the amplicons are hybridized with probes/beads, added to the amplicons, and subject to washes, where unbound DNA and debris are washed away.
- The labeled probes/beads are bound with a fluorescent label for detection and analysis.
- The product is then read by instrumentation and interpreted using software. A reviewer may have to make subjective interpretations when ambiguities exist between bead cells.
Reverse SSO is similar, but the primers and DNA are labeled and are more like a standard ELISA assay, where substrate and conjugate cause a color change or fluorescent signal for detection after DNA is bound to a medium.
