T790M<i>EGFR</i> mutant enrichment analysis by HRM and Sanger Sequencing.

<p>(A) HRM analysis plots and Sanger Sequencing chromatograms of serial dilutions of T790M <i>EGFR</i> mutations versus wild type DNA following conventional PCR. (B) The detection after mutant enrichment by conventional PNA-LNA PCR using an initial mutant abundance of 1% and 0.1% T790M, c.2369C>T. (C) Improved detection after mutant enrichment from two rounds of DISSECT followed by conventional PNA-LNA PCR from an initial mutant abundance of 1% and 0.01% T790M, c.2369C>T.</p

Schematic of procedure.

<p>(A) Flowchart diagram of <i>EGFR</i> exon 20 mutant enrichment by DISSECT followed by PNA-LNA PCR. (B) Schematic design of <i>EGFR</i> exon 20 primers, LNA probe, and PNA clamp during PNA-LNA PCR procedure.</p

Cell lines used in the present study.

<p>Cell lines used in the present study.</p

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<div><p>Non-small cell lung cancer (NSCLC) patients treated with small molecule <i>EGFR</i> inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on <i>EGFR</i> exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, <u>Di</u>fferential <u>S</u>trand <u>Se</u>paration at <u>C</u>ritical <u>T</u>emperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M <i>EGFR</i> mutation. DISSECT pre-enriches low-abundance T790M <i>EGFR</i> mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.</p></div

PCR thermocycling conditions utilized in the present work.

1<p>Conditions from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Milbury4" target="_blank">[22]</a>.</p

Temperature-tolerant-fast-COLD-PCR in emulsion: Overview of the steps involved.

<p>Multiplex pre-amplification from genomic DNA; emulsification with gene-specific primers; mixing into a single tube; and temperature-tolerant emulsion-based fast-COLD-PCR.</p

Primer sequences used in this study.

1<p>Oligonucleotides (F) forward or (R) reverse.</p>2<p>Oligonucleotide sequences described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Fredriksson1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Milbury4" target="_blank">[22]</a>.</p>3<p>Oligonucleotides from Castellanos et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-CastellanosRizaldos1" target="_blank">[16]</a>.</p>4<p>Primer sequences described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Li1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Milbury3" target="_blank">[17]</a>.</p

Comparison of PNA-LNA sensitivity of detection for the T790M<i>EGFR</i> mutation.

<p>(A) Real-time PCR plot represents serially diluted PCR product with T790M mutation (1%, 0.1% and 0.01%) into amplified product from wild type DNA, by conventional PNA-LNA PCR. (A, right) Corresponding plot of the log concentration of T790M mutant DNA versus threshold cycle number. Graph shows mutant T790M serial dilution following conventional PNA-LNA PCR. (B) Real-time PCR plot shows an increased level of detection when PNA-LNA is applied to a sample that has undergone two rounds of mutant enrichment by DISSECT. (B, right) Corresponding graph shows mutant T790M serial dilution following PNA-LNA PCR after DISSECT.</p

Temperature-tolerant COLD-PCR in emulsion, TT-fast-eCOLD-PCR: Enrichment of mutations in multiple DNA sequences in a single tube.

<p>A 5% mutation abundance was evaluated for <i>TP53</i> gene exons 6–9 by conventional PCR (left panels) and TT-fast-eCOLD-PCR (right panels). Duplicate experiments are depicted in each case. The enrichment of the mutations in all four exons is estimated from the chromatograms.</p

Melting profiles of bisulfite-converted DNA from clinical samples following <i>fast</i>-COLD-MS-PCR.

<p>The effect of lowering the denaturation temperature in PCR is depicted. <b>Panel A.</b> Glioma sample no. 3 was subjected to different critical denaturation temperature-T_c during <i>fast</i>-COLD-MS-PCR. The modulation of the preferential amplification of the un-methylated DNA fraction is shown. <b>Panel B. </b><i>fast</i>-COLD-MS-PCR performed at a T_c of 84°C demonstrates that the amplification of the methylated DNA fraction is completely inhibited.</p