Related Experiment Video
Updated: Jun 1, 2026
14:16
Fluorescence detection methods for microfluidic droplet platforms
Published on: December 10, 2011
Quantitative and sensitive detection of rare mutations using droplet-based microfluidics.
Deniz Pekin1, Yousr Skhiri, Jean-Christophe Baret
1Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 allée Gaspard Monge, BP 70028, F-67083 Strasbourg Cedex, France.
Lab on a Chip
|May 20, 2011
Summary
This study introduces a highly sensitive digital PCR method using microfluidics to detect rare cancer DNA mutations. The technique accurately quantifies mutant genes, even with a 200,000-fold excess of normal DNA, enabling precise cancer biomarker analysis.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Somatic mutations in tumoral DNA serve as specific biomarkers for cancer detection, diagnosis, prognosis, and treatment monitoring.
- Existing methods like TaqMan assays and pyrosequencing lack the sensitivity to detect rare mutant DNA (below 1%) in complex biological samples.
- A highly sensitive and quantitative method is needed to measure mutant-to-wild-type gene ratios for early and accurate cancer detection.
Purpose of the Study:
- To develop a novel procedure for highly sensitive and quantitative detection of mutated DNA within complex mixtures.
- To establish a digital PCR-based method for precise measurement of mutant allelic specific imbalance (MASI).
- To enable parallel screening of multiple KRAS mutations in a single experiment.
Main Methods:
- Utilized a droplet-based microfluidic system to perform digital PCR, compartmentalizing genomic DNA (gDNA) into millions of picolitre droplets.
- Employed two TaqMan probes (mutant-specific and wild-type specific) generating distinct fluorescent signals (green and red) within each droplet.
- Determined the mutant-to-wild-type gene ratio by counting fluorescent droplets post-thermocycling; enabled parallel screening via droplet fusion with mutation-specific probes.
Main Results:
- Demonstrated accurate and sensitive quantification of mutated KRAS oncogene in gDNA.
- Successfully determined MASI in various cancer cell lines.
- Achieved precise quantification of mutated KRAS in the presence of a 200,000-fold excess of unmutated KRAS genes.
- Showcased the ability to screen six common KRAS codon 12 mutations in parallel using droplet fusion technology.
Conclusions:
- The digital PCR microfluidic system offers unprecedented sensitivity for detecting rare mutant DNA, limited only by the number of droplets analyzed.
- This technique provides a powerful tool for sensitive and quantitative analysis of cancer biomarkers, including MASI.
- The parallel screening capability significantly enhances the efficiency of analyzing multiple mutations in cancer diagnostics.
