But real-time PCR has its drawbacks such as the need for an external reference for quantification and a lack of sensitivity at low concentrations. Until now, real-time PCR is considered as the “gold standard” for gene detection and quantification. A decade later, real-time PCR also termed quantitative PCR, offered the possibility of monitoring the PCR process. Since the invention of PCR by Kary Mullis in 1983, it has been a real revolution in molecular biology. The primer and probe sequences we used for this assay can be found in 1 C圓-labeled hydrolysis probe to detect EGFR T790M mutation.1 FAM-labeled hydrolysis probe to detect EGFR WT sequence.1 primer set to amplify the EGFR T790 locus.Then you will need for this specific experiment: A reference dye if necessary (please check instrument manufacturer’s recommendations).A PCR mastermix (please check instrument manufacturer’s recommendations) containing all the essential components to perform an experiment, including:.A digital PCR system and its dedicated consumables.In addition to all the tools you would require for a standard PCR experiment (microfuge tubes, vortex, microcentrifuge, pipettes, and tips, etc.), for a digital PCR experiment you will need : What do I need to perform a rare mutation detection experiment? PCR assay using two fluorescent probes specific for the wild-type and the mutant allele respectivelyĢ. Before ordering your probes, please check that the fluorophores you want to use are compatible with your digital PCR system in terms of excitation and emission spectra.įinally, according to the principles of the hydrolysis probes, you will observe the generation of a fluorescent signal when the target sequence (mutant or wild-type) is present.įigure 1. Then one probe, labeled with a specific fluorophore (represented as a solid blue circle in figure 1), will target the wild-type, and the other one, labeled with another specific fluorophore (represented as a solid green circle in figure 1), will target the mutant allele.
These primers will amplify the region of interest. The option we have chosen here is to use two different hydrolysis probes (TaqMan®), with only one set of primers. How should I design an assay to detect a rare mutation?įirst of all, note that the rules for designing probes and primers are the same in qPCR and digital PCR. Thus, early detection of the appearance of this mutation is of clinical importance in directing the patient to a more effective treatment. In patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC), the presence of the mutation EGFR T790M renders the standard treatment, based on first and second-generation tyrosine kinase inhibitors (TKIs), ineffective. The EGFR T790M mutation is rarely detected during the initial tumor characterization and will, most often, become detectable over the course of treatment with TKI.
We choose here to focus on the detection of mutations occurring on the epithelial growth factor receptor ( EGFR) gene. In this tutorial, we will help you to prepare and analyze a rare mutation detection experiment from A to Z. Indeed, digital PCR will enable you to detect and quantify rare mutations, even when present in very low amounts: less than 1% or even 0.1% of the wild-type (WT) sequence. Thus, for the detection and quantification of rare events, such as point mutations or single nucleotide polymorphism (SNP), digital PCR is the right tool for you! The major advantages of digital PCR are its precision, sensitivity, and accuracy.
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