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<p>Droplet Digital PCR (ddPCR) is a method for performing digital PCR that is based
on water-oil emulsion droplet technology. A sample is fractionated into 20,000 droplets,
and PCR amplification of the template molecules occurs in each individual droplet.
ddPCR technology uses reagents and workflows similar to those used for most standard
TaqMan probe-based assays. The massive sample partitioning is a key aspect of the
ddPCR technique.</p>
<p>This section provides an overview of droplet digital PCR technology, how ddPCR
works, and what the advantages and benefits of ddPCR are.</p>
<p><strong>Related topics</strong>: <a href="/evportal/destination/solutions?catID=MDV33OKG4">Planning
Droplet Digital PCR Experiments</a>, <a href="/evportal/destination/solutions?catID=MDV359ESH">Absolute
Quantification of PCR Targets with the Droplet Digital™ PCR System</a></p>
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What is Droplet Digital PCR?<p>Droplet Digital PCR technology is a digital PCR method utilizing a water-oil emulsion
droplet system. Droplets are formed in a water-oil emulsion to form the partitions
that separate the template DNA molecules. The droplets serve essentially the same
function as individual test tubes or wells in a plate in which the PCR reaction takes
place, albeit in a much smaller format. The massive sample partitioning is a key aspect
of the ddPCR technique.</p>
<p>The Droplet Digital PCR System partitions nucleic acid samples into thousands of
nanoliter-sized droplets, and PCR amplification is carried out within each droplet.
This technique has a smaller sample requirement than other commercially available
digital PCR systems, reducing cost and preserving precious samples.</p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/sample-partitioning-is-key-to-droplet-digital-pcr.jpg"
alt="Sample partitioning is the key to Droplet Digital PCR" width="429" height="243"
/></p>
<p><strong>Sample partitioning is the key to droplet digital PCR. </strong>In traditional
PCR, a single sample offers only a single measurement, but in Droplet Digital PCR,
the sample is partitioned into 20,000 nanoliter-sized droplets. This partitioning
enables the measurement of thousands of independent amplification events within a
single sample.</p>
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How Does Droplet Digital PCR Work?<p>ddPCR technology uses a combination of microfluidics and proprietary surfactant
chemistries to divide PCR samples into water-in-oil droplets (<a href="#Hindson">Hindson
et al. 2011</a>). The droplets support PCR amplification of the template molecules
they contain and use reagents and workflows similar to those used for most standard
TaqMan probe-based assays. Following PCR, each droplet is analyzed or read to determine
the fraction of PCR-positive droplets in the original sample. These data are then
analyzed using<strong> <a href="/evportal/destination/solutions?catID=MDV359ESH#Poisson">Poisson
statistics</a> </strong>to determine the target DNA template concentration in the
original sample.</p>
<a href="/evportal/destination/solutions?catID=MDV359ESH#Poisson"></a>
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Advantages of ddPCR over Other Digital PCR Technologies<p>Droplet Digital PCR surpasses the performance of earlier digital PCR techniques
by resolving the previous lack of scalable and practical technologies for digital
PCR implementation. Serial dilution is laborious and introduces the possiblity of
pipetting error; competing chip-based systems rely on complex fluidics schemes for
partitioning. Droplet Digital PCR addresses these shortcomings by massively partitioning
the sample in the fluid phase in one step. The creation of tens of thousands of droplets
means that a single sample can generate tens of thousands of data points rather than
a single result, bringing the power of statistical analysis inherent in digital PCR
into practical application. Bio-Rad's Droplet Digital PCR System automates the ddPCR
workflow of droplet generation, thermal cycling, droplet reading, and data analysis,
making this technology accessible to the working research laboratory.</p>
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The Benefits of Droplet Digital PCR<p>ddPCR technology enables high-throughput digital PCR in a manner that uses lower
sample and reagent volumes and reduces overall cost compared with other methods while
maintaining the sensitivity and precision that are the hallmarks of digital PCR.</p>
<p>The benefits of ddPCR technology include:</p>
<ul>
<li><strong>Absolute quantification</strong> — ddPCR technology provides an
absolute count of target DNA copies per input sample without the need for running
standard curves, making this technique ideal for measurements of target DNA, viral
load analysis, and microbial quantification</li>
<li><strong>Unparalleled precision </strong> — the massive sample partitioning
afforded by ddPCR enables the reliable measurement of small fold differences in target
DNA sequence copy numbers among samples</li>
<li><strong>Increased signal-to-noise ratio</strong> — high-copy templates and
background are diluted, effectively enriching template concentration in target-positive
partitions, allowing for the sensitive detection of rare targets and enabling a ±10%
precision in quantification</li>
<li><strong>Removal of PCR bias</strong> — error rates are reduced by removing
the amplification efficiency reliance of qPCR, enabling the detection of small (1.2-fold)
differences</li>
<li><strong>Simplified quantification</strong> — neither calibration standards
nor a reference (the <em>ΔΔCq</em> method) is required for absolute quantification</li>
<li><strong>Reduced consumable costs</strong> — reaction volumes are in the
pico- to nanoliter ranges, reducing reagent use and the sample quantity required for
each data point</li>
<li><strong>Lower equipment costs</strong> — the emulsion-based reaction system
means that the PCR reactions can be performed in a standard thermal cycler without
complex chips or microfluidics</li>
<li><strong>Superior partitioning </strong>— ddPCR technology yields 20,000
droplets per 20 µl sample, nearly two million partitioned PCR reactions in a
96-well plate, whereas chip-based digital PCR systems produce only hundreds or thousands
of partitions. The greater number of partitions yields higher accuracy</li>
</ul>
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QX200™ Droplet Digital™ PCR System<p>Bio-Rad’s <a href="/en-us/product/qx200-droplet-digital-pcr-system">QX200
Droplet Digital PCR (ddPCR) System</a> consists of two instruments, the QX200 Droplet
Generator and the QX200 Droplet Reader, plus their associated software and consumables.
The QX200 Droplet Generator partitions samples (20 µl into 20,000 nanoliter-sized
droplets) for PCR amplification. Following amplification using a thermal cycler, droplets
from each sample are analyzed individually on the QX200 Droplet Reader, where PCR-positive
and PCR-negative droplets are counted to provide absolute quantification of target
DNA in digital form.</p>
<p>The ddPCR System can be used to:</p>
<ul>
<li>Detect rare DNA target copies with unmatched sensitivity</li>
<li>Determine copy number variation with unrivaled accuracy</li>
<li>Measure gene expression levels with exquisite precision</li>
</ul>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/qx100-droplet-digital-pcr-system.jpg"
alt="QX100 Droplet Digital PCR system" width="340" height="179" /></p>
<p class="caption"><strong>QX200 Droplet Digital PCR System. </strong>QX200 Droplet
Reader (left) and the QX200 Droplet Generator (right).</p>
<div class="top"><a href="#helptop">Back to Top</a></div>
QX200 Droplet Digital PCR System Workflow<p>This 3-D animated video describes the principles and process of ddPCR using Bio-Rad's
Droplet Digital PCR System.</p>
<p><iframe src="http://www.youtube.com/embed/Qwma-1Ek-Y4?version=3&rel=0&showinfo=0&theme=light&modestbranding=1&fs=1;wmode=transparent"
width="597" height="336"></iframe></p>
<p>The workflow is quite simple. First, the QX200 Droplet Generator partitions samples
into thousands of nanoliter-sized droplets. After PCR on a thermal cycler, droplets
from each sample are streamed in single file on the QX200 Droplet Reader to count
positive and negative reactions. The PCR-positive and PCR-negative droplets are counted
to provide absolute quantification in digital form. The steps are described in more
detail below.</p>
<p><strong>Step 0: Prepare PCR-Ready Samples</strong><strong> prior to Starting ddPCR</strong></p>
<p><strong><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-prepare-pcr-ready-samples-prior-to-starting-ddpcr-step0.jpg"
alt="Step 0: Prepare PCR-Ready Samples prior to Starting ddPCR" width="465" height="335"
/></strong></p>
<p><strong>Step 1: Droplet Generation</strong> <br /> <br /> Prior to droplet generation,
nucleic acid samples (DNA or RNA) are prepared as they are for any real-time assay:
using primers, fluorescent probes (TaqMan probes with FAM and HEX or VIC), and a proprietary
supermix developed specifically for droplet generation. Samples are then placed into
the QX200 Droplet Generator, which utilizes proprietary reagents and microfluidics
to partition the samples into 20,000 nanoliter-sized droplets. The droplets created
by the QX200 Droplet Generator are uniform in size and volume.</p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-droplet-generation-step1.jpg"
alt="Step 1: Droplet Generation" width="465" height="335" /></p>
<p><strong>Step 2: PCR Amplification of Droplets</strong></p>
<p>Droplets are transferred to a 96-well plate for PCR amplification in any compatible
<a href="/evportal/destination/product?catID=75f1b406-3746-4580-a998-74245b094f56">thermal
cycler</a>.</p>
<p><strong><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-amplification-of-droplets-step2.jpg"
alt="Step 2: PCR Amplification of Droplets" width="465" height="302" /></strong></p>
<p><strong>Step 3: Droplet Reading</strong></p>
<p>Following PCR amplification of the nucleic acid target in the droplets, the samples
are placed in the QX200 Droplet Reader, which analyzes each droplet individually using
a two-color detection system (set to detect FAM and either HEX or VIC), enabling multiplexed
analysis for different targets in the same sample. The droplet reader and its bundled
QuantaSoft™ software count the PCR-positive and PCR-negative droplets.</p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-droplet-reading-step3.jpg"
alt="Step 3: Droplet Reading" width="465" height="322" /></p>
<table border="0" width="597">
<tbody>
<tr>
<td><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-digital-droplet-reading.jpg"
alt="Digital droplet reading" width="175" height="171" />
<p class="caption"> </p>
<p class="caption"><strong> </strong></p>
<p class="caption"><strong>Digital droplet reading.</strong> Fluorescence measurements
for each droplet in two optical channels <br />are used to count the numbers of positive
and negative droplets per sample.</p>
</td>
<td valign="top">
<p class="caption"><strong> </strong></p>
<p class="caption"> </p>
</td>
</tr>
</tbody>
</table>
<p><strong> </strong></p>
<p><strong>Step 4: Analyze Results</strong></p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-analyze-results-step4.jpg"
alt="Step 4: Analyze Results" width="465" height="299" /></p>
<p class="caption"><strong>Positive droplets, containing at least one copy of the
target, exhibit increased fluorescence over negative droplets.</strong> In ddPCR,
the QuantaSoft software measures the numbers of droplets that are positive and negative
for each fluorophore (for example, FAM and HEX) in a sample. The fraction of positive
droplets is then fitted to a Poisson distribution to determine the absolute initial
copy number of the target DNA molecule in the input reaction mixture in units of copies/µl.</p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-sample-results-from-ddpcr-experiment.jpg"
alt="Sample results from a ddPCR experiment" width="468" height="314" /></p>
<p class="caption"><strong>Sample results from a ddPCR experiment.</strong> Each droplet
in a sample is plotted on a graph of fluorescence intensity versus droplet number.
All positive droplets (those above the threshold intensity indicated by the red line)
are scored as positive, and each is assigned a value of 1. All negative droplets (those
below the threshold) are scored as negative, and each is assigned a value of 0 (zero).
This counting technique provides a digital signal from which to calculate the starting
target DNA concentration by a statistical analysis of the numbers of positive and
negative droplets in a given sample.</p>
<p>Droplet Digital PCR data from a multiplex experiment in which two targets are PCR
amplified can also be viewed as a 2-D plot in which FAM fluorescence is plotted versus
HEX fluorescence for each droplet. Because the DNA distribution into the droplets
follows a random pattern, the droplets are clustered into four groups:</p>
<ul>
<li>FAM-negative, HEX-negative</li>
<li>FAM-positive, HEX-negative</li>
<li>FAM-negative, HEX-positive</li>
<li>FAM-positive, HEX-positive</li>
</ul>
<br /> <!--
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-2d-plot-of-droplet-fluorescence.jpg"
mce_src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-2d-plot-of-droplet-fluorescence.jpg"
alt="2-D plot of droplet fluorescence" width="597" height="271" /></p>
<p class="caption"><b>2-D plot of droplet fluorescence.</b> Droplets are plotted for
Channel 1 fluorescence versus Channel 2 fluorescence.</p>
--> <img style="margin-bottom: 15px;" src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-mutation-detection.jpg"
alt="Mutation Detection" width="293" height="246" />
<p class="caption"><strong>2-D plot of droplet fluorescence.</strong></p>
<br />
<p>The QuantaSoft software fits the fraction of positive droplets to a Poisson distribution
to determine the absolute starting copy number in units of copies/µl input sample
and then reports the target DNA concentration in the form of copies per µl in
the sample.</p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-concentration-results2.jpg"
alt="Concentration results" width="503" height="275" /></p>
<p class="caption"><strong>Concentration results.</strong> Sample concentrations are
plotted as copies per µl.</p>
<p class="caption"> </p>
<p><strong>Step 5: Visualize Data</strong></p>
<p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/digital_pcr/technology_detail/digital-pcr-visualize-data-step5.jpg"
alt="digital-pcr-visualize-data-step5" width="465" height="398" /></p>
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References<ul>
<li>Bizouarn F <a href="http://www.genengnews.com/gen-articles/digital-pcr-improving-nucleic-acid-quantification/4093/"
target="_blank" rel="noopener noreferrer">http://www.genengnews.com/gen-articles/digital-pcr-improving-nucleic-acid-quantification/4093/</a></li>
<li><a name="Hindson"></a>Hindson BJ et al. (2011). High-throughput droplet digital
PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22): 8604–8610.</li>
<li>Pinheiro LB et al. (2012). Evaluation of a droplet digital polymerase chain reaction
format for DNA copy number quantification. Anal Chem 84, 1003–1011</li>
<li>Third Generation PCR (<a href="http://www.bioradiations.com/focus-on-technology/62-pcr/1329-third-generation-pcr">http://www.bioradiations.com/focus-on-technology/62-pcr/1329-third-generation-pcr</a>)</li>
<li>Droplet Digital PCR Opens New Perspectives in HIV Research (<a href="http://www.bioradiations.com/focus-on-technology/783-ddpcr/1356-droplet-digital-pcr-opens-new-perspectives-in-hiv-research-">http://www.bioradiations.com/focus-on-technology/783-ddpcr/1356-droplet-digital-pcr-opens-new-perspectives-in-hiv-research-</a>)</li>
<li>A Simple Method for Obtaining Absolute Quantification of DNA Molecules using the
Innovative Droplet Digital PCR Technology (<a href="http://www.bioradiations.com/focus-on-technology/783-ddpcr/1340-simpleddpcr">http://www.bioradiations.com/focus-on-technology/783-ddpcr/1340-simpleddpcr</a>)</li>
<li>A New Paradigm for Precise Quantitation of RNA (<a href="http://www.bioradiations.com/focus-on-technology/783-ddpcr/1333-ddpcr">http://www.bioradiations.com/focus-on-technology/783-ddpcr/1333-ddpcr</a>)</li>
<li>Sykes PJ et al. (1992). Quantitation of targets for PCR by use of limiting dilution.
Biotechniques 13: 444–449. PMID: 1389177</li>
</ul>
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6450/templatedata/internet/documentation/data/LSR/Literature/6450.xml6237/templatedata/internet/documentation/data/LSR/Literature/6237.xmlQX100 Droplet Digital PCR System Brochure, Rev C6237/webroot/web/pdf/lsr/literature/Bulletin_6237.pdfLiteraturePDFBrochures_and_SpecificationsNoQX100 Droplet Digital PCR System Brochure, Rev C62376311/templatedata/internet/documentation/data/LSR/Literature/6311.xmlQX200 Droplet Digital PCR System Brochure, Rev C6311H/webroot/web/pdf/lsr/literature/Bulletin_6311.pdfLiteraturePDFBrochures_and_Specifications/webroot/web/images/general/icons/icon_pdf.gifQX200 Droplet Digital PCR System BrochureNoQX200™ Droplet Digital™ PCR System Brochure, Rev C63116311, amplification, pcr, digital pcr, droplet digital, droplet pcr, dpcr, ddpcr,
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detection, rare mutation detection, rare sequence detection, red, rmd, rsd, rt-ddpcr,
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6407/templatedata/internet/documentation/data/LSR/Literature/6407_1393524927.xmlDroplet Digital PCR Applications Guide6407/webroot/web/pdf/lsr/literature/Bulletin_6407.pdfLiteraturePDFBrochures_and_SpecificationsDroplet Digital PCR Applications GuideNoDroplet Digital PCR Applications Guide64076407, assay design, CNV, copy number variation, data analysis, ddPCR, ddPCR statistics,
ddPCR workflow, digital technology, DNA sequencing, gene expression analysis, HER2,
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transcription ddPCR, 186-3001, 1863001, 186-4001, 1864001, application
6554/templatedata/internet/documentation/data/LSR/Literature/6554_1399586432.xml6277/templatedata/internet/documentation/data/LSR/Literature/6277.xmlProbing Copy Number Variations Using Bio-Rad's QX100 Droplet Digital PCR System, Rev
A
6277/webroot/web/pdf/lsr/literature/bulletin_6277.pdfLiteraturePDFProduct_Information_SheetsNo62776451/templatedata/internet/documentation/data/LSR/Literature/6451_1387577658.xmlDroplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital
PCR System Application Note, Rev A
6451/webroot/web/pdf/lsr/literature/Bulletin_6451.pdfLiteraturePDFApplication_Notes/webroot/web/images/general/icons/icon_pdf.gifDroplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital
PCR System Application Note, Rev A
NoDroplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital
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6578/templatedata/internet/documentation/data/LSR/Literature/6578_1411685676.xmlDroplet Digital PCR: Multiplex Detection of <em>KRAS</em> Mutations in Formalin-Fixed,
Paraffin-Embedded Colorectal Cancer Samples Application Note
6578H/webroot/web/pdf/lsr/literature/Bulletin_6578.pdfLiteraturePDFApplication_Notes/webroot/web/images/general/icons/icon_pdf.gifDroplet Digital PCR: Multiplex Detection of <em>KRAS</em> Mutations in Formalin-Fixed,
Paraffin-Embedded Colorectal Cancer Samples Application Note
NoDroplet Digital PCR: Multiplex Detection of <em>KRAS</em> Mutations in Formalin-Fixed,
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6679/templatedata/internet/documentation/data/LSR/Literature/6679_1418173918.xmlDroplet Digital PCR: Multiplex Screening of <em>KRAS</em> Mutations in Cell-Free DNA
Colorectal Cancer Samples Application Note, Ver B
6679/webroot/web/pdf/lsr/literature/Bulletin_6679.pdfLiteraturePDFApplication_NotesDroplet Digital™ PCR: Multiplex Screening of <em>KRAS</em> Mutations in Cell-Free
DNA Colorectal Cancer Samples Application Note, Rev A
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Karen MossWhat is Droplet Digital PCR?Droplet Digital PCR (ddPCR) uses nanodroplet sample partitioning for exquisitely sensitive
and economical DNA quantification without standard curves.
pcr, digital pcr, droplet digital pcr, ddpcr, droplet pcr, dpcr, ddpcr assay, screening,
quantification, copy number, mutation, qx100, qx200, autodg, quantalife, raindance
11/27/12 10:31 AM11/28/22 12:28 AMAE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA,VNenLSR/LSR/Technologies/Digital_PCRN00Introduction to Digital PCR
/en-us/applications-technologies/droplet-digital-pcr-ddpcr-technology?ID=MDV300E8Z
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