Application Notes 2018-02-21T11:34:19+00:00

Application notes

Quantifying EGFR Mutations
with Multiplex Crystal dPCR

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Detecting activating and resistance EGFR mutations in a single assay

In non-small cell lung cancer (nsclc), the epidermal growth factor receptor (EGFR) is an important therapeutic target. EGFR activating mutations, such as L858R and L861Q are predictive of disease respondiveness to targeted therapy using tyrosine kinase inhibitors (TKIs). On the contrary, the presence of EGFR T790M mutation is associated with tumor resistance to TKIs.

To monitor patient response to TKIs treatment with a simple assay, a multiplex digital PCR assay targeting EGFR L858R, L861Q and T790M, as well as wild-type EGFR was developed. These mutations were successfully and reliably detected in a background of wild-type DNA at concentrations down to 0.25 copies per microliter (final concentration), representing a mutant allele frequency of 0.05%.

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Crystal Digital PCR
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, and can be detected using the same filters as those used for fluorescein.

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.

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Droplet recovery
With  Crystal Digital PCR

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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.
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