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A specific exon 20 mutation (T790M) was also detected by length analysis of fluorescently labeled (FAM) PCR products on a capillary electrophoresis device (ABI 3100 Avant, Applied Biosystems, Foster City, California, United States), based on a new NlaIII restriction site created by the T790M mutation (2369 C→T), using the following primers: EGFR Ex20F, 5′-FAM- CTCCCTCCAGGAAGCCTACGTGAT-3′ and EGFR Ex20R 5′- TTTGCGATCTGCACACACCA-3′.
For genotyping of the COMT Val to Met change at position 108 as described in [ 91 ] , Forward: 5'-ACTGTGGCTACTCAGCTGTG-3' and Reverse 5'-CCTTTTTCCAGGTCTGACAA-3' primers were used followed by restriction digestion with NlaIII.
Presence of the mutant 2369 T nucleotide creates a new NlaIII restriction digest site, yielding a slightly shorter product (97 bp), readily detected by fluorescent capillary electrophoresis.
After PCR amplification with exon-20-specific primers spanning nucleotide 2369, wild-type sequence contains specific NlaIII sites, which upon digestion yield a 106-bp product (see Methods; Figure 3A).