Supplementary Fig.1

Genomic DNA

EGFR exon 20 ssDNA isolated by Streptavidin Magnetic Beads

Fragmented genome

Restriction endonuclease digestion

Hybridization with biotinylated probetargeting EGFR exon 20

PNA-enriched TaqMan real-time PCR to detect T790M mutation

A

PNA clamp inhibiting wtB

TaqMan probe targeting T790MForward primer

Reverse primer

Biotinylated probe

3’ RsaI RsaI 5’

B

Minus strand

XLNA

Supplementary Fig. 1. Outline of PNA-clamp-based TaqMan real-time PCR detection of T790M using isolated EGFR exon 20 targets. (A) RsaI-digested genomic DNA is hybridized with biotinylated EGFR exon 20 probe, followed by magnetic beads purification of EGFR exon 20 single strand DNA. A real-

time PCR with PNA clamp probe that inhibits the amplification of wild-type DNA and a TaqMan probe with a centrally located locked-nucleic acid nucleotide matching the mutation is used for real-time detection of T790M mutation from the purified single strand DNA of EGFR exon 20. (B) Schema for

the design of probe and primers.