supplementary fig.1
DESCRIPTION
A. Genomic DNA. Restriction endonuclease digestion. Fragmented genome. Hybridization with biotinylated probe targeting EGFR exon 20. EGFR exon 20 ssDNA isolated by Streptavidin Magnetic Beads. PNA-enriched TaqMan real-time PCR to detect T790M mutation. B. B. - PowerPoint PPT PresentationTRANSCRIPT
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.