Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Summary

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Abstract

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Introduction

Thousands of somatic mutations related to cancer development have been reported¹. Among these, a few are predictive markers of the efficacy of targeted therapy ^2,3 and genetic screening of these mutations is now routine clinical practice. Droplet digital PCR (ddPCR) technology can be used to monitor for the presence or the absence of mutations with high detection accuracy and is highly compatible with non-invasive liquid biopsies^4,5. However, current ddPCR assays are primarily designed to detect mutations known a priori....

Protocol

The protocol presented here follows the ethics guidelines of the Institut Curie. All human samples were obtained from patients enrolled, after informed consent, in studies approved by the Institutional Review Board at Institut Curie.

1. Blood Collection, Plasma Storage and Cell-free DNA Extraction

NOTE: DNA extracted from any type of “tissue” can be used (e.g., fresh or formaldehyde-fixed paraffin embedded (FFPE) tissues, cells in culture or blood samples). Here, we provide detailed instructions for blood collection, plasma isolation and storage, and cell-free DNA (cfDNA) extraction.

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Results

In a proof-of-concept study, KRAS exon 2 mutations (codons 12 and 13) and EGFR exon 19 deletions were investigated in FFPE tissues and plasma samples from cancer patients using the drop-off ddPCR strategy⁶.

The KRAS drop-off probe interrogated a 16 bp region encompassing multiple mutations in exon 2 of the KRAS gene, which harbor more than 95% of the known KRAS

Discussion

To design an efficient drop-off ddPCR assay, optimization is crucial, and the protocol must be followed carefully. Each combination of primers and probes has a unique PCR reaction efficiency. Thus, an individual assay has to be carefully validated on control samples before being used on valuable test samples. Optimization and validation are important to certify peak signal detection and assess specificity and sensitivity. As described in the protocol, all mutations located in a target region covered by the "probe 2&#.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
K2EDTA tube (color code : lavender)	BD	367863	EDTA tubes are used to obtain a whole blood or EDTA plasma sample
QIAamp Circulating Nucleic Acid Kit (manual protocol)	Qiagen	55114	For isolation of free-circulating DNA from human plasma
QIAsymphony DSP Circulating DNA Kit (automated protocol)	Qiagen	937556	For isolation of free-circulating DNA from human plasma
QIAsymphony SP system	Qiagen	9001297	fully integrated and automated system for cfDNA, DNA or RNA purification
QIAvac 24 Plus	Qiagen	19413	For purification of up to 24 cfDNAs simultaneously
Qubit fluorometer	Invitrogen	Q33226	The Qubit fluorometer is a benchtop fluorometer for the quantitation of DNA
QX100 or QX200 reader	Bio-Rad	186-3003 or186-4003, respectively	The reader measures fluorescence intensity of each droplet and detects the size and shape as droplets pass the detector
QX100 or QX200 droplet generator	Bio-Rad	186-3002 or 186-4002, respectively	Instrument used for droplet generation
C1000 thermal cycler	Bio-Rad	1851196	Modular thermal cycler platform, includes C1000 Touch thermal cycler chassis, 96-well fast reaction module
Plate sealer	Bio-Rad	181-4000	PX1™ PCR plate sealer
DG8 cartridge holder	Bio-Rad	186-3051	Positions and holds the DG8 cartridge in the instrument for droplet generation
Droplet generator cartridges and gaskets	Bio-Rad	186-4007	Microfluidic DG8 cartridge used to mix sample and oil to generate droplets; DG8 gaskets seal the cartridge to prevent evaporation and apply the pressure required for droplet formation
PCR supermix	Bio-Rad	186-3010	ddPCR supermix for probes (no dUTP)
96-well PCR plates	Eppendorf	951020362	96-well semi-skirted plates
Foil seal	Bio-Rad	181-4040	Pierceable foil heat seal
ddPCR Droplet Reader Oil	Bio-Rad	186-3004	oil used in the read
Droplet Generation Oil for Probes	Bio-Rad	186-3005	oil for droplet generation
DNA loBind Tube 1.5 mL	Eppendorf	0030 108.051	Eppendorf LoBind Tubes maximize sample recovery by significantly reducing sample-to-surface binding
Multiplex I cfDNA Reference Standard Set	HORIZON	HD780	The Multiplex I cfDNA Reference Standards are highly-characterized, biologically-relevant reference materials used to assess the performance of cfDNA assays that detect somatic mutations
QUBIT DSDNA HS ASSAY KIT, 500	Life Technologies	Q32854	The HS assay is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to be accurate for initial sample concentrations from 10 pg/µL to 100 ng/µL
Qubit Assay Tubes	Life Technologies	Q32856	Qubit assay tubes are 500 µL thin-walled polypropylene tubes for use with the Qubit Fluorometer