BLAST isn’t “free” when you consider hidden costs such as lost time and the higher reagent costs due to the need to redesign primers and probes.
Trial and error primer and probe synthesis and optimization with BLAST can become expensive due to a few core limitations:
- Hits are scored on sequence similarity rather than thermodynamic affinity.
- Simulation is not possible under actual experimental oligonucleotide concentrations and salt conditions.
- False-amplicons and off-target effects cannot be quantified.
- DNA/DNA, RNA/RNA, or DNA/RNA hybrid duplexes cannot be properly scored for basepair matches or mismatch geometry.
- Oligonucleotide secondary structure such as bulges, gaps, hairpins and dangling ends are completely ignored.
- Modified nucleotides and backbones are completely ignored.