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Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Abstract : Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan® assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan®probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRASgene in the presence of a 200 000-fold excess of unmutated KRASgenes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan®probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAScodon 12 in parallel in a single experiment.
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https://hal.archives-ouvertes.fr/hal-02148770
Contributor : Jean-Christophe Baret Connect in order to contact the contributor
Submitted on : Wednesday, June 5, 2019 - 6:03:21 PM
Last modification on : Monday, December 13, 2021 - 1:10:03 PM

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Deniz Pekin, Yousr Skhiri, Jean-Christophe Baret, Delphine Le Corre, Linas Mazutis, et al.. Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab on a Chip, Royal Society of Chemistry, 2011, 11 (13), pp.2156. ⟨10.1039/C1LC20128J⟩. ⟨hal-02148770⟩

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