ULTRASENSITIVE DETECTION AND CHARACTERIZATION OF CLUSTERED KRAS MUTATIONS USING PEPTIDE NUCLEIC ACID CLAMP PCR IN DROP-BASED MICROFLUIDICS

摘要

This disclosure employs the combination of a microfluidics platform and drop-based digital polymerase chain reaction (dPCR) to create a breakthrough technology that enables the detection of CTC. genes and the isolation of single CTCs from the blood. In the first method, cDNA molecules from lysed CTCs are amplified in microfluidic drops and detected via fluorescence signal. In the second method, intact single CTCs are encapsulated, and amplification-positive drops are sorted from the remaining cells. To demonstrate the clinical utility of our technology, mutations in the KRAS gene in colorectal cancer are analyzed to study resistance to EGFR-based treatment as a test case. The methods herein present robust techniques for both the diagnosis and treatment of cancers, as well as for the obtainment of a pure CTC sample from billions of other cells in the blood.