Table 2.
Method | Features |
---|---|
Microdissection | Allows precise analysis of tumor tissue by reducing contamination with normal tissue and, therefore, improving sensitivity for RAS mutations present in the neoplastic cell sample |
COLD PCR | Exploits the critical temperature at which mutation-containing DNA is preferentially melted over WT (sensitivity, 0.01–10 %) |
Allele-specific amplification | Uses primers designed with a 3′ terminal nucleotide that pairs with the mutant sequence but not with the WT (e.g., the amplification refractory mutation system) (sensitivity, 0.1–1 %) |
Blocking amplification of WT sequence | For example, by binding high-affinity peptide nucleic acid probes (PCR clamp; sensitivity in routine applications, 0.1–1 %) |
Selective destruction of WT samples | Exploiting restriction enzyme sites in the WT sequence (e.g., restriction endonuclease-mediated selective PCR and simultaneous PCR/restriction fragment length polymorphism) (sensitivity, 0.001–0.1 %) |
COLD co-amplification at lower denaturation temperatures, PCR polymerase chain reaction, WT wild-type