Detection and quantitation of FGFR3 mutations (p.R248C, p.S249C, p.G370C and p.Y373C) and corresponding wild type sequences were performed by droplet digital PCR (ddPCR), using the QX200 system (Bio-Rad Laboratories, Hercules, CA) and hydrolysis probe-based assays (PrimePCR ddPCR Mutation Detection Assays; Bio-Rad). The PCR mixture contained 11 μl of ddPCR droplet supermix for probes (no dUTPs), 1.1 μl of mutation primer/probe mix (FAM), 1.1 μl of wild type primer/probe mix (HEX) and 2 μl of DNA in a final volume of 22 μl. Twenty microliters of this mixture and 70 μl of droplet generation oil were transferred to different wells of a droplet generation cartridge. After formation of droplets using the droplet generator, samples were transferred to a 96-well PCR plate and subjected to amplification for 40 cycles at 94°C for 30 sec. and 55°C for 60 sec. Droplets (on average ~16,000 per reaction) were analyzed on the droplet reader, and Quantasoft software (version 1.4.0.99) was used for analyzing DNA concentrations. Cutoff settings were determined using mutation-positive and-negative control DNA samples. For urine samples processed both by filtration and sedimentation, equimolar amounts of DNA were used as template.
Primepcr ddpcr mutation detection assay
Manufactured by Bio-Rad
Sourced in United States
PrimePCR ddPCR Mutation Detection Assays are a suite of pre-designed and validated assays for the detection of specific gene mutations using the droplet digital PCR (ddPCR) technique. These assays provide a sensitive and accurate method for quantifying the presence of target mutations in DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Quantasoft v 1, by Bio-Rad (3 mentions)
Qx200 system, by Bio-Rad (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (1 mentions)
Qx200 droplet digital pcr, by Bio-Rad (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qiacube, by Qiagen (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Qx200 ddpcr system, by Bio-Rad (1 mentions)
4 protocols using primepcr ddpcr mutation detection assay
1
Droplet Digital PCR for FGFR3 Mutation Detection
2
Detecting KRAS Mutations in Liquid Biopsy
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DNA was extracted from plasma and peritoneal fluid with the QIAamp Circulating Nucleic Acid Kit and the DNeasy Blood & Tissue kit was used for cell lines (Qiagen). The starting volume was 3 ml for plasma and 2 ml for peritoneal fluid. ddPCR analyses were performed using the QX200 Droplet Digital PCR System (Bio-Rad). KRAS G12D, G12V and G13D mutations were detected with PrimePCR™ ddPCR™ Mutation Detection Assays (Bio-Rad). DNA from LS-174T, SW480 and HCT-116 human adenocarcinoma cell lines was used as a positive control of these mutations, respectively. SW480 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). LS-174T and HCT-116 were kindly provided by the Translational Oncology Division, OncoHealth Institute. KRAS wild-type control DNA was obtained from healthy donor peripheral blood mononuclear cells. No-template controls (NTCs) adding water instead of DNA to the reaction mixture were also included. Results were analyzed using Quantasoft v.1.7 software (Bio-Rad). Two to four replicates of each sample were analyzed.
3
Droplet Digital PCR for BRAF V600E Mutation Detection
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DNA was extracted on a Qiacube semiautomated robotic device (Qiagen, Valencia, CA) using either the QIAamp DNA Mini Kit (Qiagen) from 17 FNA washout samples and 6 frozen tissue samples, or the QIAamp DNA FFPE Tissue Kit (Qiagen) for paraffin-embedded tissue sections, according to the instructions of the manufacturer. BRAF T1799A (V600E) mutational analysis was performed using the PrimePCR ddPCR mutation detection assay (BIO-RAD, Hercules, CA) on a BIO-RAD QX200 droplet digital PCR (ddPCR) system. Each reaction included 10 μl of 2x ddPCR supermix for probes (no dUTP), 1 μl of BRAF V600E primer/probe mix (FAM), 1 μl of BRAF wild type primer/probe mix (HEX), and 40–100 ng of genomic DNA. The presence of mutation and the fractional abundance of the mutant allele was determined with QuantaSoft v.1.7 (BIO-RAD).
4
Quantification of ctDNA by ddPCR
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Cell-free DNA (cfDNA) was isolated from 2–4 mL plasma samples using the MagMax cell-free DNA isolation kit with the KingFisher Prime Duo instrument (ThermoFisher) according to the manufacturer’s instructions. Circulating tumor DNA (ctDNA) detection was performed on a BIO-RAD QX200 ddPCR system using the custom PrimePCR ddPCR mutation detection assay (BIO-RAD, Hercules, CA, USA) for specific EGFR mutations originally identified in a patient’s tumor specimen (Supplementary Table S2 ). Each PCR reaction contained 10 µL of 2× ddPCR supermix for probes (no dUTP), 1 µL 20× mutant primers/probe mix (FAM) and wild type primers/probe mix (HEX) mix, 1 µL nuclease-free water, and 8 µL of cfDNA. The assay was performed in duplicate. The presence of mutant DNA copies and the fractional abundance of the mutant allele were determined with QuantaSoft v.1.7 (BIO-RAD). Mutant EGFR copy number was normalized to plasma volume and is expressed as copies/mL plasma. ctDNA progression by ddPCR was defined using the following criteria: (1) conversion from negative to positive AFs, or (2) increase in AFs two consecutive timepoints by more than 10%.
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