Principle of the IDH1/2 PCR assay. Total reaction mixes (top) The total Primers and Probe Mixes (PPM-Total) used primers and probes to amplify both mutated and wild-type (WT) target sequences. Mutation detection reaction mixes (middle) The mutation detection primers and probe mixes combined primers and probes, to amplify both mutated and WT target sequences, plus an oligonucleotide, 3'-blocked with the addition of a phosphate group (3’-Oligo-P) to prevent elongation (PCR clamping), which was specific to the WT target sequence. When the PCR template contained the WT sequence, the 3'-Oligo-P bound preferentially over the PCR primer binding due to higher affinity. There was no or low extension by the DNA polymerase and no or low amplification was observed. When a mutated sequence was present, PCR primer bound preferentially over the 3'-Oligo-P and amplification proceeded. Mutation identification reaction mixes (bottom) Allele-specific amplification was achieved by ARMS® (Amplification Refractory Mutation System), which exploits the ability of the DNA polymerase to distinguish between a match and a mismatch at the 3' end of a PCR primer. When the PCR primer fully matched, the amplification proceeded with full efficiency. When the 3' base was a mismatch, only low-level background amplification occurred. The same principle shown on the figure to detect IDH1 R132H applied for IDH1 R132C and IDH2 R172K.