Posts filed under ‘Droplet Digital PCR’

Everything You Need to Know About Droplet Digital PCR

ddpcr guideDroplet Digital polymerase chain reaction (ddPCR™) was developed to provide high-precision, absolute quantification of nucleic acid target sequences with wide-ranging applications for both research and clinical diagnostic applications. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions. Droplet Digital PCR using Bio-Rad’s QX100™ or QX200™ Droplet Digital PCR system overcomes the previous lack of scalable and practical technologies for digital PCR implementation.

ddPCR has the following benefits for nucleic acid quantification:

  • Unparalleled precision — the massive sample partitioning afforded by ddPCR enables small fold differences in target DNA sequence between samples to be reliably measured
  • Increased signal-to-noise — enrich for rare targets by reducing competition that comes from high-copy templates
  • Removal of PCR efficiency bias — error rates are reduced by removing the amplification efficiency reliance of PCR, enabling accurate quantification of targets
  • Simplified quantification — a standard curve is not required for absolute quantification

Download the Droplet Digital™ PCR Applications Guide for a comprehensive explanation of this powerful technique.

The guide includes chapters on:

  1. Designing Droplet Digital™ PCR Experiments
  2. Absolute Quantification and the Statistics of Droplet Digital™ PCR
  3. Copy Number Variation Analysis
  4. Rare Mutation and Sequence Detection
  5. Gene Expression
  6. Next-Generation Sequencing Library Analysis
  7. Droplet Digital™ PCR Tips, Assay Considerations, and Troubleshooting

November 25, 2014 at 3:23 pm Leave a comment

Droplet Digital™ PCR Tips & Tricks: ddPCR™ Assay Design

August 26, 2014 at 7:12 am Leave a comment

Introduction to Droplet Digital™ PCR: Workflow and Applications

August 11, 2014 at 4:15 pm Leave a comment

Using Droplet Digital™ PCR for Cancer and Liquid Biopsy Studies

January 27, 2014 at 4:11 pm Leave a comment

Droplet Digital™ PCR Works for GMO Quantification

A study published in PLOS ONE finds that Droplet Digital PCR (ddPCR™) technology is suitable for routine analysis of genetically modified organisms (GMOs) in food, feed, and seeds.

More than 60 countries representing 40 percent of the world’s population require labeling of food and feed when GMOs reach certain thresholds. Screening for and quantifying GMOs is essential to the integrity of this labeling policy.

“Droplet Digital PCR could replace or be a good alternative to qPCR, the current benchmark in GMO quantification,” said Dr. Dany Morisset, the paper’s lead author and a researcher at Slovenia’s National Institute of Biology. Dr. Morisset, in collaboration with the EU Reference Laboratory for GM Food and Feed (EU-RL GMFF), also coordinates an international R&D project to standardize screening methods for detecting GMOs in food and feed.

The paper showed that Droplet Digital PCR (ddPCR™) technology is more accurate and reliable than real-time quantitative PCR (qPCR) for quantifying GMOs, especially at low levels. Study authors also found that the ddPCR method meets international food standards of applicability and practicality.

qPCR has Drawbacks for Detecting GMOs
The most common technique for quantifying the presence of GMOs is qPCR, thanks to its accuracy and precision. However, according to Dr. Morisset, qPCR has several drawbacks. It is often unreliable and inaccurate when quantifying very small numbers of DNA targets or when those targets are part of complex matrices such as foods or feed that contain inhibitory substances.

A 2010 research study found that chamber digital PCR (cdPCR) delivered accurate quantification at low target copy number without the need for a standard curve. The matrix also did not inhibit cdPCR because it is an end-point assay and therefore its data are less affected by amplification efficiency. However, Dr. Morisset says its high costs make cdPCR impractical for real-world use.

The ddPCR System Meets or Exceeds International Recommendations for Performance Parameters
Dr. Morisset learned about Droplet Digital PCR technology, which was developed as an alternative to cdPCR with its easy workflow, low cost, and high throughput. Commercialized as the QX100™ Droplet Digital PCR system the ddPCR system provides thousands more partitions than in cdPCR, resulting in greater precision and per-sample costs that are up to 150 times less.

The Slovenian researchers analyzed food and feed matrices containing different percentages of a well-characterized GMO transgene. They found the ddPCR system’s performance parameters (precision, accuracy, sensitivity, and dynamic range) complied with the guidelines of the EU-RL GMFF and were comparable or superior to those for qPCR. Compared with the conventional qPCR assay, the ddPCR assay offered better accuracy at low target concentrations and greater tolerance to inhibitors found in matrices such as wheat flour and feed.

ddPCR Technology is Practical for Everyday Lab Use
International food safety standards specify that new methods should be easy for labs to implement in terms of cost, time, and workflow.

In the authors’ hands, a ddPCR assay requires 190 minutes and a qPCR assay takes 160 minutes for the typical number of samples run in parallel in midsize GMO laboratories. However, due to the greater number of PCR reactions required per sample in the qPCR assay, the time and expense of the qPCR assay grows rapidly with increasing sample throughput. Droplet Digital PCR is also simpler to set up and involves less hands-on labor than qPCR.

Dr. Morisset’s findings reveal that ddPCR is a less expensive alternative to qPCR due to the lower number of reactions. Droplet Digital PCR capitalizes on its ability to duplex as opposed to qPCR’s traditional approach of performing separate assays for both control and transgene targets. The ddPCR assay also doesn’t require reactions for a standard curve or dilutions due to lower anticipated inhibition.

May 13, 2013 at 4:05 pm Leave a comment

Finding the rare mutant allele needle in the wild-type haystack

Studies of biomarkers associated with the onset of and progression of cancers have been fraught with poor selectivity and have failed to detect mutant sequences with abundances of less than 1 in 100 wild-type sequences. The QX100 ddPCR platform offers a simple and robust means to increase the precision and sensitivity of TaqMan assays for rare event detections. Not only are direct counts of template molecules within a sample possible (Pinheiro et al. 2012), but partitioning mitigates the effects of target competition, greatly improving the discriminatory capacity of assays that differ by only a single nucleotide. Here, the benefits for clinically relevant assay targets are demonstrated through the BRAF V600E mutation assay, where the LOD improved >100-fold when performed in ddPCR. This platform should prove invaluable to the molecular diagnostics field in general and to the cancer field in particular for the routine assessment of mutation status. Combined with the high accuracy achieved through the use of tens of thousands of partitions, ddPCR represents a unique platform for extremely precise and sensitive measurements of target molecules.

Click here to learn more on how Bio-Rad Laboratories’ QX100™ Droplet Digital™ PCR System can aid in the detection of rare mutant alleles within a background of wild-type sequences.

October 4, 2012 at 4:05 pm Leave a comment

The ultimate tool for quantitative DNA measurement

The QX100™ Droplet Digital™ PCR (ddPCR™) system provides an absolute measure of target DNA molecules with unrivaled performance in precision, accuracy, and sensitivity for quantitative PCR applications. The Droplet Digital PCR system is the third generation of PCR technology and provides a revolutionary approach to target DNA quantification.

Watch the video below for a fantastic explanation.

May 27, 2012 at 1:17 pm Leave a comment

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