ddPCR Mutation Detection Assays

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ddPCR Mutation Detection Assays

Overview

QX200™ Droplet Digital™ PCR System Brochure

Droplet Digital PCR (ddPCR™) is Bio-Rad's unique digital PCR technology.

  • Unrivaled precision provides absolute quantification of target DNA or RNA molecules without the use of standard curves
  • Addresses the lack of scalable and practical technologies for digital PCR implementation

The QX200 ddPCR system makes this powerful technology accessible to you for unveiling previously unattainable
research levels.

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QX200™ Droplet Digital™ (ddPCR™) System Data
Detection of BRAF V600E Mutation

High sensitivity of the QX200 system allows quantification of BRAF V600E mutation in the presence of wild-type DNA using PrimePCR ddPCR assays. A, 2-D fluorescence amplitude plot generated by QuantaSoft™ software shows triplicate wells of a mixed mutant:wild-type sample. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for wild-type DNA only, the blue cluster represents the droplets that are positive for mutant DNA only, and the orange cluster represents the droplets that are positive for both mutant and wild-type DNA. B, 2-D fluorescence amplitude plot shows three replicates of a wild-type-only sample. C, fractional abundance plot shows the percentage frequency (orange markers) of the mutant DNA in a wild-type DNA background. The blue markers indicate the concentration of mutant DNA (copies/µl) and the green markers indicate the concentration of wild-type DNA (copies/µl) in each of three replicate samples. All error bars generated by QuantaSoft software represent the 95% confidence interval.



Detection of Herpes Simplex Virus and β2 Microglobulin

ddPCR enables precise and reproducible detection of herpes simplex virus 1 (HSV-1) and ß2 microglobulin (B2M) targets in a duplex assay. A, 2-D fluorescence amplitude plot shows three merged replicate samples of 10,000 copies/well of HSV-1 duplexed with 20,000 copies/well of Β2M. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for HSV-1 only, the blue cluster represents the droplets that are positive for B2M only, and the orange cluster represents the droplets that are positive for both HSV-1 and B2M targets. B, concentration plot shows merged triplicate wells across an HSV-1 sample dilution series from 0 to 40,000 copies in a constant background of 20,000 copies (66 ng human genomic DNA) of B2M. The blue markers indicate HSV-1 copies/µl and the green markers indicate B2M copies/µl. All error bars generated by QuantaSoft software represent the 95% confidence interval.



Detection of Copy Number for
Myelocytomatosis Oncogene and β-Globin

The QX200 system provides superior resolution with copy number (CN) calls from CN1 to CN13. A, 2-D fluorescence amplitude plot shows duplicate wells of a CN6 sample duplexed with myelocytomatosis oncogene (MYC) and β-globin (HBB) assays. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for MYC only, the blue cluster represents the droplets that are positive for HBB only, and the orange cluster represents the droplets that are positive for both MYC and HBB targets. B, copy number plot shows samples ranging from CN1 to CN13 with precise duplicate well values. All error bars generated by QuantaSoft software represent the 95% confidence interval. NTC, no template control.



Validation for Next-Generation Sequencing

ddPCR provides nearly 1,000-fold greater sensitivity and an amplification bias–free approach compared to RNA-Seq for validation. A, concentration plot shows a twofold dilution of cDNA generated using Bio-Rad’s iScript™ advanced cDNA synthesis kit for RT-qPCR. Two independent measurement sets were made: one at a high concentration range and one at a lower range, with four points overlapping. B, table shows comparative results between ddPCR and MiSeq. ddPCR detected thousands of copies per well with low-abundance transcripts (TBP and GUSB genes) using an input of 100 ng total RNA, which demonstrates enhanced sensitivity. The MiSeq sequencer detected only single-digit reads per kilobase per million reads (RPKM) for these transcripts. ddPCR, Droplet Digital PCR.



Gene Expression Analysis of Stem Cell Markers

High sensitivity and precision of the QX200 system allow reliable detection of small fold changes with rare transcripts. The TGFβ signaling pathway plays a role in ovarian stem cell regulation, and the addition of TGFβ will increase the number of stem cells in the population, increase the expression of putative stem cell markers, and decrease terminal differentiation markers like E-cadherin (ECAD). Data show expression levels of cKIT, CD133, OCT3/OCT4, and ECAD genes across ten samples with and without TGFβ treatment, normalized to PPIA in this study. Reproducible detection of cDNA copies as low as 0.5–1 target/µl in a 20 µl reaction was achieved. S, sample.


Digital PCR using QX200™ and AutoDG™ ddPCR Systems
Success Stories: Articles and Videos

Success Stories: Articles

Genomic Variation Analysis: Single Nucleotide Polymorphism: Validation of Allele-Specific Expression Predicted by RNA-Seq in Human Brain Specimens
In the context of human brain development, the QX100™ Droplet Digital™ PCR System yields unambiguous results for genotype identification of low-expression genes and provides absolute quantification of allele-specific expression (ASE) without the use of a standard.
Download the article.

Genomic Variation Analysis: Copy Number Variation: DUF1220 Copy Number Variation across Healthy and Diseased Populations
Studying copy number variation (CNV) with the QX100 Droplet Digital PCR System allows the robust identification of genetic factors such as DUF1220 in neurogenetic diseases between individuals with greater accuracy and precision, while effectively validating array CGH data.
Download the article.

Genomic Variation Analysis: Single Nucleotide Polymorphism: Detection of Single Nucleotide Variation Frequencies by Droplet Digital™ PCR
The QX100 Droplet Digital PCR System validated DNA sequencing results of induced pluripotent stem cells (iPSCs). In addition to confirming the high frequency of single nucleotide variations (SNVs), a de novo type of SNV was identified.
Download the article.

Cancer Mutation Analysis: How Droplet Digital PCR Has Revolutionized Noninvasive Plasma-Based Detection of Mutations
Performing Droplet Digital PCR with the QX100 Instrument effectively detected and accurately quantified the EGFR T790M marker in targeted therapies with limited cancer tumor samples.
Download the article.

Viral Quantification: Adeno-Associated Virus Vector Genome Titer Assay
Determining the adeno-associated virus (AAV) vector genome (vg) titer assay is critical in gene therapy applications. The improved sensitivity observed with Droplet Digital PCR (ddPCR™) technology makes it highly suitable for titration of clinical-grade AAV vector preparations.
Download the article.

Gene Expression: Digital Quantification of Potential Therapeutic Target RNAs
Measuring the exact number of long noncoding RNAs (lncRNAs) in subcellular compartments with Droplet Digital PCR (ddPCR) provides insights into their role and mechanism of action.
Download the article.

Third Generation PCR: A Novel Way to Accurately Measure Copy Number Variations in Human Genome
The McCarroll Lab uses Droplet Digital technology to study the distribution and molecular properties of the genome and its influence on gene expression, as well as the genetics underlying schizophrenia and bipolar disorder.
Download the article.

Success Stories: Videos

Droplet Digital™ PCR opens new perspectives in HIV research. Sangamo BioSciences, Inc. is developing HIV therapeutics toward a functional cure using the company’s proprietary zinc finger DNA-binding protein technology.
Download the article.


Professor Alec Morley, a pioneer in digital PCR, and his colleagues in Adelaide have developed tests for detecting acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) using the unrivaled sensitivity of the QX200 Droplet Digital PCR System.



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Droplet Digital PCR Publications

Browse the growing list of cited peer-reviewed journal articles in Droplet Digital PCR research featuring Bio-Rad products.

Browse Publications

ddPCR Mutation detection assays can be found and purchased on Bio-Rad.com/digital-assays. A wet-lab validated mutation detection assay is returned if available, or an assay is designed instantly for your target. Wet-lab validated mutation detection assays detect mutant DNA present at 0.1% in a background of WT DNA in one well. An even better sensitivity of 0.005–0.1% can generally be achieved by using multiple reaction wells. Browse list of wet-lab validated Mutation Detection Assays.

Mutation detection assays can be designed instantly for over 4 million mutations, listed in the COSMIC database, on Bio-Rad’s Digital Assay Site. Built by the experts in digital PCR at Bio-Rad's Digital Biology Center, the Bio-Rad Digital Assay Site design engine takes into consideration everything from GC content and Tm, to primer placement and probe length, even genome specificity when designing an assay for you.

Key Benefits

  • Wet-lab validated assays available for more than 200 targets
  • Wet-lab validated assays guaranteed to work when used as instructed
  • Validation data available for review and download
  • Assays designed instantly for targets and sequences for which validated assays are not available
  • Suitable for use with circulating tumor DNA, liquid biopsy, and FFPE and other tissue samples
  • Orderable in single-tube or two-tube formats
  • Positive control sequences provided

Sample Data




High sensitivity of PrimePCR ddPCR mutation detection assays provide precise quantification of JAK2 V617F mutation in the presence of wild-type DNA. A, 2-D fluorescence amplitude plot shows duplicate wells of a mixed mutant: wild-type sample across multiple percentage mutant blends. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for wild-type DNA only, the blue cluster represents the droplets that are positive for mutant DNA only, and the orange cluster represents the droplets that are positive for both wild-type and mutant targets. B, 2-D fluorescence amplitude plot shows duplicate wells of a wild-type–only sample. C, fractional abundance plot shows the percentage frequency (orange markers) of the mutant DNA in a wild-type DNA background. All error bars are generated by QuantaSoft software and represent the 95% confidence interval.

Packaging Options

ddPCR Mutation Detection Assays are available in single tube or two-tube formats.

Single-tube format

The single-tube formats of the ddPCR Mutation Detection Assays contain a set of primers, a mutant, and wild-type probe. The single tube format assays are 20X concentrated, ready-to-use-primer-probe mix optimized for use with ddPCR Supermix for Probes (no dUTP). Each assay contains 18 uM each primer and 5uM each probe. The single-tube formats provide added convenience as the mutant and WT probes are pre-mixed and allow for the addition of an extra sample in the ddPCR reaction.

Catalog# Product Name Reaction Size
10049550 ddPCR MUT FAM+HEX Assay, Validated 200
10049551 ddPCR MUT FAM+HEX Assay, Validated 1000
10049552 ddPCR MUT FAM+HEX Assay, Validated 2500
 
10049047 ddPCR MUT FAM+HEX Assay 200
10049048 ddPCR MUT FAM+HEX Assay 1000
10049049 ddPCR MUT FAM+HEX Assay 2500

Two-tube formats

The mutant and WT probes are provided separately.

Catalog# Product Name Reaction Size
10031246 ddPCR MUT FAM Assay, Validated 200
10031247 ddPCR MUT FAM Assay, Validated 1000
10031248 ddPCR MUT FAM Assay, Validated 2500
10031249 ddPCR MUT HEX Assay, Validated 200
10031250 ddPCR MUT HEX Assay, Validated 1000
10031251 ddPCR MUT HEX Assay, Validated 2500
 
10042964 ddPCR MUT FAM Assay 200R 200
10042965 ddPCR MUT FAM Assay 1000R 1000
10042966 ddPCR MUT FAM Assay 2500R 2500
10042967 ddPCR MUT HEX Assay 200R 200
10042968 ddPCR MUT HEX Assay 1000R 1000
10042969 ddPCR MUT HEX Assay 2500R 2500

Related Products

Resources

Number Description Options
7144 Wet-Lab Validated ddPCR Assays for Mutation Detection and Copy Number Determination Brochure Click to download
10000050420 ddPCR Mutation Detection Assays, Validated, Product Insert Click to download
10000050422 ddPCR Mutation Detection Assays Product Insert Click to download
10033487 ddPCR Mutation Detection Assays, Validated, Product Insert Click to download
10047489 ddPCR Mutation Detection Assays Product Insert Click to download

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