Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutations at codons 12 and 13 in KRAS, and 2 mutations (L858R, and T790M) in EGFR in clinical samples. The real-time Bi-PAP could detect 0.01% mutation in the presence of 100 ng template DNA. Of the 34 samples from the colon cancer patients, real-time Bi-PAP detected 14 KRAS mutant samples whereas the traditional real-time allele-specific PCR missed two samples with mutation abundance <1% and DNA sequencing missed nine samples with mutation abundance <10%. The detection results of the two EGFR mutations in 45 non-small cell lung cancer samples further supported the applicability of the real-time Bi-PAP. The real-time Bi-PAP also proved to be more efficient than the real-time allele-specific PCR in the detection of templates prepared from formalin-fixed paraffin-embedded samples. Thus, real-time Bi-PAP can be used for rapid and accurate quantification of somatic mutations. This flexible approach could be widely used for somatic mutation detection in clinical settings

Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

<div><p>Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutations at codons 12 and 13 in <i>KRAS</i>, and 2 mutations (L858R, and T790M) in <i>EGFR</i> in clinical samples. The real-time Bi-PAP could detect 0.01% mutation in the presence of 100 ng template DNA. Of the 34 samples from the colon cancer patients, real-time Bi-PAP detected 14 <i>KRAS</i> mutant samples whereas the traditional real-time allele-specific PCR missed two samples with mutation abundance <1% and DNA sequencing missed nine samples with mutation abundance <10%. The detection results of the two <i>EGFR</i> mutations in 45 non-small cell lung cancer samples further supported the applicability of the real-time Bi-PAP. The real-time Bi-PAP also proved to be more efficient than the real-time allele-specific PCR in the detection of templates prepared from formalin-fixed paraffin-embedded samples. Thus, real-time Bi-PAP can be used for rapid and accurate quantification of somatic mutations. This flexible approach could be widely used for somatic mutation detection in clinical settings.</p></div

Working principle of real-time Bi-PAP.

<p>The working principle is schematically illustrated for the detection of a mutant template (shown as GC-containing, left panel) using a pair of Bi-PAP primers and a molecular beacon. The four steps: primer annealing, pyrophosphorolysis, primer extension/probe hybridization, and fluorescence detection, are shown from the top to bottom. As a result, fluorescence generated from the probe hybridization is detected in the form of amplification profile. In case of a wild-type template (shown as TA-containing, right panel), two Bi-PAP primers cannot be activated and extended owing to 3′-terminal mismatch, leading to no probe hybridization and thus no fluorescence signal.</p

Study on the sensitivity and specificity of the singleplex real-time Bi-PAP.

<p>(A) Amplification curves of 5000, 500, 50, 5 copies per reaction (from the left to right) of G12R mutant plasmids. (B) Amplification curves of 500, 100, 10, 1 ng wild-type genomic DNA per reaction. Water was used as non-template control (NTC).</p

Quantitative performance of the two-color duplex real-time Bi-PAP.

<p>(A) Amplification curves of 100%, 10%, 1%, 0,1%, 0.01%, and 0% G12R mutant (from left to right) in the presence of 100 ng wild-type genomic DNA (upper panel). The linear relationship of the Cq difference between the mutation and the internal control (ΔCq =  Cq – Cq^IC) with respect to the logarithmic mutation percentages (lower panel). (B) Amplification curves of the internal control with different mutant percentages (upper panel). The linear relationship between the Cq values of the internal control and the logarithmic mutant percentages (lower panel).</p

The limit of detection of the two-color duplex real-time Bi-PAP for each KRAS mutation in the presence of 100 ng wild-type genomic DNA.

<p>For each mutation, the ΔCq values for wild-type (red), 0.01% mutant (blue), and 0.1% (black) were detected in 10 replicates and calculated. The line within the box denotes the median, the square within the box denotes the mean, the horizontal borders of each box denote the SD, and the whiskers denote the minimum and maximum.</p

Performance comparison of real-time Bi-PAP and real-time ARMS PCR in the detection of template DNA derived from frozen tissue and FFPE tissue samples.

<p>(A) Real-time Bi-PAP. (B) TheraScreen <i>EGFR</i> RGQ PCR. (C) AmoyDx ARMS <i>EGFR</i>. Amplification curves shown are from 20 frozen tissue samples (black lines) and 25 FFPE tissue samples (red lines), respectively. The difference between the average amplification Cq values (indicated with a double-headed arrow) from the two types of samples are given for each assay in the detection of the internal control.</p

Quantitative performance of the three-color triplex real-time Bi-PAP.

<p>Amplification curves of 50%, 10%, 1%, 0.1%, 0.01%, and 0% mutant (from left to right) are shown for L858R (A) and T790M (B) in the upper panel. The linear relationship of the Cq difference between the mutation and the internal control (ΔCq =  Cq – Cq^IC) with respect to the logarithmic mutation percentages are shown in the lower panel. Amplification curves of the internal control (C) with different mutant percentages are shown in the upper panel. The linear relationship between the Cq values of the internal control and the logarithmic mutant percentages is given in the lower panel.</p

High-Density Dispersion of Atomic Pt (Ru, Rh, Pd, Ir) Induced by Meso-Stable Penta-Coordinated Fe^III in the Topological Transformation of Layered Double Hydroxides

Atomically dispersed metal sites afford high activity or selectivity in many heterogeneous catalytic reactions. It is still an open challenge to achieve a high-density dispersion of atomic metals. Herein, this work demonstrates a facile strategy to boost the dispersion density of atomic metals by the induction of meso-stable lattice distortion sites in the topological transformation of layered double hydroxides (LDHs). Meso-stable penta-coordinated FeIII, resulting from the difference in the thermal stability between Mg–OH and Fe–OH in a LDH lattice, is utilized from MgFe–LDHs as anchoring sites for atomic Pt. The dispersion density of atomic Pt reaches 2.0 Pt1/nm2. This strategy in the topological transformation of LDHs has been successfully extended to prepare high-density atomic Ru, Ir, Pd, and Rh. In both catalytic oxidation of HCHO and hydrogenation of furfuryl alcohol, high-density atomic Pt affords high turnover frequency (TOF) by the simultaneous high-efficiency activation of multimolecules on adjacent atomic Pt sites. In HCHO oxidation, high-density atomic Pt affords high mass activity under a high concentration, high space velocity, and low temperature. In furfuryl alcohol hydrogenation, high-density atomic Pt affords high mass activity while retaining >99% selectivity of 2-methylfuran