Digital PCR
Application notes
Drop-Off Crystal™ dPCR
for NRAS, KRAS, & EGFR mutations
for NRAS, KRAS, & EGFR mutations
How to design and quantify using a drop-off assay
A major advantage of a 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 most simplified
version of a drop-off assay includes two TaqManTM 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
(Figure 1A). In the presence of a wild-type allele, both the drop-off and reference probes will hybridize with their
target, leading to a double positive signal (Figure 1B, turquoise population). 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 1B, green population)
3-Color Crystal™ Digital PCR assays
for EGFR mutation detection
for EGFR mutation detection
Two multiplex digital PCR assays detect EGFR activating and resistance mutations
In non-small cell lung cancer, the epidermal growth factor receptor (EGFR) is a common therapeutic target. EGFR activating mutations, such as L858R, L861Q and exon 19 deletions are predictive of disease responsiveness to targeted therapy using tyrosine kinase inhibitors (TKIs) [1]. On the contrary, the presence of EGFR T790M mutation is associated with tumor resistance to TKIs [2].
To detect and quantify these mutations in single tests without sacrificing the precision and reliability of the results, two multiplex assays were developed. Primer and probes using three different fluorescence channels of the Naica System™ enable detection of EGFR L858R/L861Q and T790M in panel 1 and EGFR exon 19 deletions (as a drop-off assay) and T790M in panel 2. Both panels also detect wild-type (WT) EGFR.
Detection of HER2 Copy Number Variation
Detecting Triplex Crystal Digital PCR assay to detect HER2 Copy Number Variation
Investigating HER2 amplification status is crucial in breast cancer to determine the indication of anti-HER2 targeted therapy. Assessing HER2 copy number variation is challenging especially when tumor DNA is diluted in DNA from healthy cells. Here, we illustrate how Crystal Digital PCR is capable of reliably identifying HER2 amplification in samples with low tumoral fraction.
An assay previously described for the detection of HER2 (ERBB2) and MRM1 on chromosome 17, and TSN (reference gene) on chromosome 2, was optimized for Crystal Digital PCR. This assay enables the quantification of HER2 copy number and the distinction between HER2 amplification and chromosome 17 polysomy, a condition that could lead to HER2 status misinterpretation.
RNA Quantification
With Crystal RT- dPCR
With Crystal RT- dPCR
Multiplexing targets with Crystal RT-dPCR
Reverse-transcription PCR (RT-PCR) is widely used for RNA analysis, for example for genomic expression or viral detection, and is applied to many fields, from life science research to diagnostics. Crystal RT-dPCR enables straightforward multiplexing of targets (references or transcripts of interest), absolute quantification of viral RNA without need for standard curves and allows exquisite detection of fine differences in gene expression.
Thanks to 3-color Crystal RT-dPCR, three different messenger RNA (mRNA) can be analyzed in a single one-step reaction, demonstrating that 1.5-fold differences in gene expression can easily and precisely be quantified.
Droplet recovery
With Crystal Digital PCR
With Crystal Digital PCR
Recovering Droplets from Sapphire Chips
The Naica System allows the recovery of droplets from the Sapphire chips with a simple procedure that can be performed before or after PCR. DNA contained in the recovered emulsion is extracted using a standard chloroform protocol and is suitable for downstream analysis using NGS, digital or real-time PCR, or gel electrophoresis. The recovery and extraction procedure takes approximately 1h for up to 24 samples.
The recovery rate for this protocol was assessed by generating a droplet crystal containing known amounts of human genomic DNA, counting droplets in the Sapphire Chips before and after recovery, extracting DNA from droplets using the protocol described above, and quantifying the amount of DNA recovered by Crystal Digital PCR. We found that:
- at least 98% of the droplets are recovered from the Sapphire Chips
- on average, 70% of the total DNA present in the initial droplet crystal is recovered.
Effect of Inhibitors
Crystal dPCR vs qPCR
Crystal dPCR vs qPCR
Discover
Quantification using real-time PCR (qPCR) vs Crystal Digital PCR
Real-time PCR quantification is based on comparing Cq values between unknown samples and standards containing known amounts of DNA. For adequate quantification, this technique therefore relies on similar PCR efficiencies between the analyzed samples and the standards. However, in presence of an inhibitory concentration of a given substance in the sample, PCR efficiency may differ between standard and sample, thus biasing quantification (Figure 1A). In Crystal Digital PCR, the droplets are characterized as either positive or negative, whereas the amplitude of fluorescence separating the two populations indicates PCR efficiency. Even in case of decreased PCR efficiency due to inhibitors, accurate quantification can still be achieved.
Crystal Digital PCR
Using EvaGreen®
Using EvaGreen®
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Setting-up EvaGreen™-based quantification in Crystal Digital PCR
EvaGreen is a non-mutagenic and non-cytotoxic DNA-binding dye compatible with Crystal Digital PCR. EvaGreen, which is non-fluorescent when free in solution, becomes strongly fluorescent upon binding in a sequence-independent manner to double-stranded DNA.
When used with Crystal Digital PCR, EvaGreen enables absolute quantification of a target by using a simple primer pair. Similarly to probe-based Crystal Digital PCR, experiments using EvaGreen will enable reliable quantification down to 0.2 copies per microliter.