Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion

Background: Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens. Methods: Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature $(T_c)$ corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion. Results: Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined. Conclusions: TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube

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

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

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

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

Cell lines used in the present study.

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

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients

EGFR Del19 screening on lung cancer patients plasma using two-plex assay.

<p>These four plots were obtained from dPCR analysis on DNA extracted from plasma of lung cancer patients. The use of Del19 castPCR™ probe permitted to screen samples containing different deletions on exon 19 (of three, four, five and six amino acids, in panel A, B, C, D, respectively). In the table, event counts from the single experiments are listed. Input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer (three μL were used for each sample). Measured allelic frequencies are given for dPCR and NGS analysis. <i>Reference</i>, <i>wild-type + mutant DNA; NA</i>, <i>not analyzed; A</i>.<i>U</i>, <i>arbitrary units; AA</i>, <i>aminoacids</i>.</p

Examples of titration series with EGFR castPCR™, ZEN™ and TaqMan® probes.

<p>Serial dilutions of L858R, T790M, Del19, or L861Q mutated DNA (extracted from H1975 cell line, H1650 cell line or FFPE tissue, respectively) in human wild-type genomic DNA. Individual data points are displayed for independent replicates. The expected mutant to wild-type ratio (black line) is shown. Green continuous and dashed lines represent LOB and LOD values, respectively, evaluated from droplets falling into the mutated-DNA cluster and analyzed in a WT gDNA sample for each replicate. For the lowest titration point (0.01%), we used a higher amount of input DNA. Thus, corresponding LOB and LOD values are represented by red lines. Since number of FP was increasing with quantity of input DNA for EGFR p.T790M castPCR™ test, LOB and LOD calculation could not be performed (refer to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159094#pone.0159094.ref008" target="_blank">8</a>]).</p