Development of novel droplet-based microfluidic strategies for the molecular diagnosis of cancer

• Main Author: Pekin, Deniz
• Language: English
• Published: Université de Strasbourg 2013
• Subjects: [SDV:AEN] Life Sciences/Food and Nutrition; [SDV:AEN] Sciences du Vivant/Alimentation et Nutrition; [SDV:SA] Life Sciences/Agricultural sciences; [SDV:SA] Sciences du Vivant/Sciences agricoles; Droplet microfluidics; Cancer biomarkers; Digital PCR
• Online Access: http://tel.archives-ouvertes.fr/tel-00856594; http://tel.archives-ouvertes.fr/docs/00/85/65/94/PDF/PEKIN_Deniz_2013_ED222.pdf

The aim of this work is to establish novel strategies for the highly sensitive screening of cancer biomarkers in biological samples.To achieve this goal, we developed droplet-based microfluidic dPCR technique. Using a limiting dilution, individual DNA molecules are encapsulated within monodisperse droplets of a water-in-oil emulsion created with a microfluidic device. Fluorescent TaqMan® probes targeting the screened cancer biomarkers allow the detection of mutations. We focused on the mutations in the human KRAS gene for the development of our test. This method is also transposable in a multiplexed format for the parallel detection of multiple mutations in clinical samples.The developed technique allowed the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of un-mutated KRAS genes and enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines. We validated our technique by screening for KRAS mutations in the blood plasma and tumor samples from patients with metastatic colorectal cancer.