Step

1. 3 3 3 3 ’ ’ ’ ’ 5 5 5 5 ’ ’ ’ ’ GTT GTT GTT GTT S S S Figure S1 . PNA-PCR/OCEAN method for K-ras mutation detection. Assymetric PNA-PCR: a PNA probe complementary to the wild type gene suppresses amplification of the codon 12 wild type K-ras sequence and amplifies preferentially mutant sequences. The PCR reaction is performed using an imbalanced forward:reverse primer ratio in order to preferentially amplify the strand which is complementary to the OCEAN probes. OCEAN: steps 1-4 are carried out in a single tube. Step 1: a stabilizing probe (anchor, in dark gray) binds to the amplified strand of K-ras; step 2: a second probe (amplifier, in light gray, carrying a Fluorescein and a Black Hole Quencher, BHQ1) complementary to the mutant gene recognizes and binds the duplex forming a three-way junction. The resulting ternary structure contains the recognition site for a restriction endonuclease (RE). Step 3: The endonuclease cleaves the amplifier. The cleavage separates the fluorophore from the Black Hole Quencher with consequent emission of fluorescence. Step 4: the cleaved amplifier dissociates, and the cycle repeats itself thus resulting in a continuous signal amplification. For each sample, 4 different OCEAN reactions are performed, each containing a different labelled amplifier, specific for the 4 most frequent mutations of K-ras codon 12. Asymmetric PNA-PCR/OCEAN for Kras mutation detection mutated mutated mutated mutated gene : : : : gene wild wild wild wild type type type type : : : : GGT 3 3 3 3 ’ ’ ’ ’ 5 5 5 5 ’ ’ ’ ’ CCA PNA: amplification amplification NO NO amplification amplification Step Step Step Step 4 4 4 4 F F F F AAC BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 BHQ1 5 5 5 5 ’ ’ ’ ’ TTG TTG TTG TTG 3 3 3 3 ’ ’ ’ ’ RE F Step Step Step Step 1 1 1 1 AAC ’ TTG TTG TTG TTG TTG TTG TTG TTG 5 3 3 3 3 3 3 3 3 ’ ’ ’ ’ ’ ’ ’ ’ Step 3 BHQ1 BHQ1 BHQ1 BHQ1 F F F F AAC 5 5 5 5 ’ ’ ’ ’ 3 3 3 3 ’ ’ ’ ’ TTG TTG TTG Step 2