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This study compares sequencing outcomes of biotin-purified templates against non-purified genomic DNA after PNA-PCR and TaqMan real-time PCR. The sequencing of biotin-purified targets demonstrated a reduction in false-positive mutations, specifically the T790M C>T mutation, which appeared even in wild-type DNA when amplification was conducted directly from genomic DNA. Results from duplicate independent experiments are presented, showcasing the superiority of target purification in avoiding PCR errors associated with non-target enriched genomic DNA.
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WT 1:1000 A. Biotin probe-isolated template B. Genomic DNA template Supplementary Fig. 3. Sequencing of biotin-purified template vs. sequencing of non-purified template, following PNA-PCR. PNA-enriched TaqMan real-time PCR products from purified targets (A) or genomic DNA (B) were subjected to sequencing with forward strand. Duplicate independent experiments are shown. The arrows indicate the position of T790M C>T mutation. When the PNA-PCR amplification is performed directly from genomic DNA (Panel B), mutations emerge even for wild-type DNA. Target purification via biotinylated probe avoids such false-positive mutations (PCR errors) compared with non-target enriched genomic DNA (Panel A). Supplementary Fig. 3
