Table 2.

Methods for enriching mutant DNA for RAS testing [49–53]

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