How to design and quantify using a drop-off assay
A major advantage of drop-off digital PCR is the single-assay detection of multiple proximal genetic lesions (including deletions, insertions and nucleotide substitutions) within a short genomic interval. The simplest version of a drop-off assay includes two TaqMan™ probes targeting the same amplicon: a drop-off probe that spans the mutation hotspot but is uniquely complementary to the wild-type sequence, and a reference probe that hybridizes adjacent to the mutation site and is complementary to both the mutant and the wild-type alleles. In the presence of a wild-type allele, both the drop-off and reference probes will hybridize with their targets, leading to a double-positive signal. By contrast, in the presence of a mutant allele, even a single nucleotide mutation is enough to destabilize the hybridization of the drop-off probe so that only the reference probe anneals to its target, leading to a simple positive signal.
Figure 1 | Reliability of drop-off assays for the detection of the seven and four most prevalent KRAS exon 12–13 and NRAS exon 3 mutations, respectively, and EGFR exon 19 deletions. The mutations were detected with a 95% confidence level in serial dilutions ranging from 5% to 0.25% mutant DNA at final concentrations down to 1 copy/μl in a 25 μl PCR mixture. All assays were performed in a reaction background of 104 copies of wild-type DNA and 400 copies of the internal positive control DNA (ΦX174 bacteriophage). N = 3 replicates for each dilution point. The displayed confidence intervals are the means of the theoretical confidence intervals accounting for sampling and partitioning error at a 95% confidence level.
Drop-off assays detect KRAS, NRAS and EGFR hotspot mutations
In clinical settings, a set of predictive genetic markers is routinely monitored to track therapy efficacy. For example, in non-small-cell lung cancer, the presence of deletions in the epidermal growth factor (EGFR) exon 19 confers sensitivity to first-generation tyrosine kinase inhibitors. Moreover, in colorectal carcinoma, KRAS and NRAS proto-oncogene mutations are strong indicators of resistance to anti-EGFR antibodies. Using drop-off assays and the three-color multiplexing capacity of the Naica system, three internally controlled drop-off digital PCR assays were designed to detect the most prevalent KRAS exon 12, NRAS exon 3 and EGFR exon 19 sequence alterations (Figs. 1 and 2). The addition of an internal control allows the straightforward evaluation of assay robustness and the identification of PCR inhibition, which can occur as a result of sample impurity.
As multiple mutations are known to occur in KRAS and NRAS hotspots and several deletions or insertions of varying lengths have been described in EGFR exon 19, the use of drop-off assays allows rapid and cost-efficient simultaneous screening of a variety of clinically relevant genetic alterations using a limited number of probes. If desired, once a drop-off assay has identified a sample as mutant, subsequent assays using sequence-specific probes can be employed to determine the exact mutant alleles existing within the DNA sample.
Figure 2 | 2D dot plots of the triplex drop-off assays. a. Crystal Minergenerated2D dot plots of the KRAS, NRAS and EGFR drop-off digital PCR assaysin triplex experiments. Commercial DNA (KRAS and NRAS), as well as DNA derived from frozen tumor samples (EGFR), was used. WT, wild type. b, Dot plots displaying the signal obtained in the third color channel for each assay using the ΦX174 DNA internal positive-control detection assay.
Application Note highlights
• Drop-off digital PCR assays enable the simultaneous detection of multiple mutations occurring at genomic hotspots.
• Drop-off assays allow rapid and cost-efficient screening of a variety of genetic alterations using a limited number of probes.
• Using Crystal Digital PCR™, we designed and validated three internally controlled drop-off assays for the detection of seven KRAS mutations, four NRAS mutations and a range of EGFR exon 19 deletions or insertions commonly monitored in clinical practice.
Nature Methods