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 KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. 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 KRAS codon 12 in parallel in a single experiment.

Detection and characterization of circulating cell free tumor DNA in cancer patients with malignant solid tumors. Liquid biopsy: a new tool in molecular pathology?

“…By counting the number of"positive" partitions (in which the sequence is detected) after PCR versus "negative" partitions (in which it is not), you can determine exactly how many copies of a DNA molecule were in the original sample. Positive reactions are labeled as "1" and negative reactions as "0", hence the term " digital" PCR [32]. By using dPCR it is possible to reach a sensitivity of >0.01% mutation load in a wild-type background.…”

DNA Nanoparticles for Improved Protein Synthesis In Vitro

“…The discrete nature of compartments enables digital quantification of absolute numbers of nucleic acids in a sample,3 accurate estimation of copy‐number variation,4 detection of pathogens5 and rare cancer mutations,6 as well as other applications 7…”

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

“…No clearly standardized procedures for KRAS/NRAS mutational testing have been proposed and established (12,13) and an increasing number of techniques have been developed with different levels of sensitivity and specificity (12,(14)(15)(16)(17)(18)(19)(20)(21). Several highly sensitive techniques are currently being evaluated.…”