Droplet Digital™ PCR (ddPCR™) Technology

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en-us MDV31M4VY Droplet Digital PCR ddPCR Technology Droplet Digital™ PCR (ddPCR™) Technology /webroot/web/html/lsr/solutions/technologies/digital_pcr <script type="text/javascript"><!-- $('head').append('<meta name="DCSext.at_banner" content="Droplet Digital PCR ddPCR Technology" /><meta name="DCSext.at_banner_e" content="v" />'); // --></script> <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&trade; PCR System</a></p> <div class="bannerAT" style="margin-left:10px;"> <div class="bannerText ddpcrText"> <h2 class="banner_header">Validated ddPCR&trade; Assays</h2> <p>Our PrimePCR&trade; Assays for Droplet Digital&trade; PCR are designed and validated for copy number variation, rare mutation detection, NGS library quantification, and high-sensitivity gene expression studies.</p> <a class="linkgeneration" href="/_locale/product/digital-pcr/digital-pcr-assays?at_banner=Droplet Digital PCR ddPCR Technology&amp;at_banner_rev=Droplet Digital PCR ddPCR Technology&amp;at_banner_e=c">Learn more &raquo;</a></div> <img src="/webroot/web/images/lsr/global/english/solutions/lab-validated-ddpcr-assays.jpg" alt="Lab-Validated ddPCR&trade; Assays" width="615" height="120" /></div> <script src="/webroot/web/js/countrySpecific-min.js" type="text/javascript"></script> <script type="text/javascript"><!-- $(document).ready(function(){ setSterlingUrlsToHtmlHrefVariables(); $('a.linkgeneration').each(function(){ $(this).attr('href', $(this).attr('href').replace('_locale', languageCode + '-' + countryCode).replace('_verticalUrl', currentVerticalUrlTitle).replace('_defaultVerticalUrl', defaultVerticalUrlTitle).replace('_feedbackCMSID',feedbackCMSID)); }); }); // --></script> 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> <div class="top"><a href="#helptop">Back to Top</a></div> 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> <div class="top"><a href="#helptop">Back to Top</a></div> 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> <div class="top"><a href="#helptop">Back to Top</a></div> 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> &mdash; 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> &mdash; 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> &mdash; 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 &plusmn;10% precision in quantification</li> <li><strong>Removal of PCR bias</strong> &mdash; 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> &mdash; neither calibration standards nor a reference (the <em>&Delta;&Delta;Cq</em> method) is required for absolute quantification</li> <li><strong>Reduced consumable costs</strong> &mdash; 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> &mdash; 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>&mdash; ddPCR technology yields 20,000 droplets per 20 &micro;l 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> <div class="top"><a href="#helptop">Back to Top</a></div> QX200&trade; Droplet Digital&trade; PCR System <p>Bio-Rad&rsquo;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 &micro;l 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&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;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&trade; 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">&nbsp;</p> <p class="caption"><strong><br /></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><br /></strong></p> <p class="caption">&nbsp;</p> </td> </tr> </tbody> </table> <p><strong><br /></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/&micro;l.</p> <strong> <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> </strong> <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/&micro;l input sample and then reports the target DNA concentration in the form of copies per &micro;l 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 &micro;l.</p> <p class="caption">&nbsp;</p> <strong> <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> </strong> <div class="top"><a href="#helptop">Back to Top</a></div> References <ul> <li>Bizouarn F <a href="http://www.genengnews.com/gen-articles/digital-pcr-improving-nucleic-acid-quantification/4093/" target="_blank">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&ndash;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&ndash;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&ndash;449. PMID: 1389177</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> 6450 /templatedata/internet/documentation/data/LSR/Literature/6450.xml 6237 /templatedata/internet/documentation/data/LSR/Literature/6237.xml QX100&#153; Droplet Digital&#153; PCR System Brochure, Rev C 6237 /webroot/web/pdf/lsr/literature/Bulletin_6237.pdf Literature PDF Brochures_and_Specifications No QX100&#153; Droplet Digital&#153; PCR System Brochure, Rev C 6237 6311 /templatedata/internet/documentation/data/LSR/Literature/6311.xml QX200™ Droplet Digital™ PCR System Brochure, Rev C 6311 H /webroot/web/pdf/lsr/literature/Bulletin_6311.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif QX200 Droplet Digital PCR System Brochure No QX200™ Droplet Digital™ PCR System Brochure, Rev C 6311 6311, amplification, pcr, digital pcr, droplet digital, droplet pcr, dpcr, ddpcr, rt-ddpcr, qx100, qx200, real-time pcr, pcr quantitation, quantitative pcr, qpcr, absolute quantification, cnv, copy number variation, digital technology, dna sequencing, gene expression analysis, mutation detection, next-generation sequencing, ngs, rare event detection, rare mutation detection, rare sequence detection, red, rmd, rsd, rt-ddpcr, 186-3001, 186-3001ja, 181-4000, 185-1197, 186-3004, 186-3005, 186-3009, 186-3010, 186-3021, 186-3022, 186-3023, 186-3024, 186-3025, 186-3026, 186-3027, 186-3028, 186-3040, 186-3041, 186-3051, 186-3052, 186-4001, 186-4002, 186-4003, 186-4005, 186-4006, 186-4007, 186-4008, 186-4033, 186-4034, 186-4035, 186-4036, 186-4052, 1863001, 1863001ja, 1814000, 1851197, 1863004, 1863005, 1863009, 1863010, 1863021, 1863022, 1863023, 1863024, 1863025, 1863026, 1863027, 1863028, 1863040, 1863041, 1863051, 1863052, 1864001, 1864002, 1864003, 1864005, 1864006, 1864007, 1864008, 1864033, 1864034, 1864035, 1864036, 1864052 6407 /templatedata/internet/documentation/data/LSR/Literature/6407_1393524927.xml Droplet Digital PCR Applications Guide 6407 /webroot/web/pdf/lsr/literature/Bulletin_6407.pdf Literature PDF Brochures_and_Specifications Droplet Digital PCR Applications Guide No Droplet Digital PCR Applications Guide 6407 6407, assay design, CNV, copy number variation, data analysis, ddPCR, ddPCR statistics, ddPCR workflow, digital technology, DNA sequencing, gene expression analysis, HER2, linkage analysis, microRNA amplification, milepost assay, mir-210 miRNA, mutation detection, next-generation sequencing, NGS, one-step RT-ddPCR kit for probes, quantification, QX100 Droplet Digital PCR system, QX200, rare event detection, rare mutation detection, rare sequence detection, RED, RMD, RSD, RT-ddPCR, troubleshooting, two-step reverse transcription ddPCR, 186-3001, 1863001, 186-4001, 1864001, application 6554 /templatedata/internet/documentation/data/LSR/Literature/6554_1399586432.xml Droplet Digital&#8482; PCR: Detection of DNA Methylation Application Note, Rev A 6554 H /webroot/web/pdf/lsr/literature/Bulletin_6554.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Droplet Digital&#8482; PCR: Detection of DNA Methylation Application Note, Rev A 6554 6554, bisulfite conversion, ddPCR technology, digital quantitation, digital technology, DNA sequencing, next-generation sequencing, NGS, qPCR, quantification, quantitative PCR, QX100, QX100 system, QX200, splice variants, splicing 6277 /templatedata/internet/documentation/data/LSR/Literature/6277.xml Probing Copy Number Variations Using Bio-Rad's QX100 Droplet Digital PCR System, Rev A 6277 /webroot/web/pdf/lsr/literature/bulletin_6277.pdf Literature PDF Product_Information_Sheets No 6277 6451 /templatedata/internet/documentation/data/LSR/Literature/6451_1387577658.xml Droplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital PCR System Application Note, Rev A 6451 H /webroot/web/pdf/lsr/literature/Bulletin_6451.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif Droplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital PCR System Application Note, Rev A No Droplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital PCR System Application Note, Rev A 6451 6451, ddPCR, ddPCR technology, digital quantitation, digital technology, DNA sequencing, multiplex assays, next-generation sequencing, NGS, PCR amplification, qPCR, quantification, quantitative PCR, QX100, QX100 system, QX200, real-time PCR, RT-ddPCR 6578 /templatedata/internet/documentation/data/LSR/Literature/6578_1411685676.xml Droplet Digital&trade; PCR: Multiplex Detection of <em>KRAS</em> Mutations in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples Application Note, Rev A 6578 H /webroot/web/pdf/lsr/literature/Bulletin_6578.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif Droplet Digital PCR: Multiplex Detection of KRAS Mutations in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples Application Note No Droplet Digital PCR: Multiplex Detection of KRAS Mutations in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples Application Note 6578 6578, cancer therapies, CRC, ddPCR technology, digital quantification, digital technology, EGFR pathway mutations, FFPE samples, genomic testing, multiplex, multiplex detection, multiplexing, qPCR, quantification, quantitative PCR, QX100, QX200, QX200 system, singleplex, therapy strategies 6679 /templatedata/internet/documentation/data/LSR/Literature/6679_1418173918.xml Droplet Digital&trade; PCR: Multiplex Screening of <em>KRAS</em> Mutations in Cell-Free DNA Colorectal Cancer Samples Application Note, Rev A 6679 /webroot/web/pdf/lsr/literature/Bulletin_6679.pdf Literature PDF Application_Notes Droplet Digital&trade; PCR: Multiplex Screening of <em>KRAS</em> Mutations in Cell-Free DNA Colorectal Cancer Samples Application Note, Rev A No Droplet Digital&trade; PCR: Multiplex Screening of <em>KRAS</em> Mutations in Cell-Free DNA Colorectal Cancer Samples Application Note, Rev A 6679 6679, cancer therapies, cfDNA samples, CRC, ddPCR technology, digital quantification, digital technology, EGFR pathway mutations, FFPE samples, genomic testing, multiplex assay, multiplex detection, multiplexing, qPCR, quantification, quantitative PCR, QX100, QX200, therapy strategies Life Science Research/Products/Amplification - PCR/Digital PCR/QX100 Droplet Digital PCR ->MTS::M9HE3XE8Z##Life Science Research/Products/Amplification - PCR/Thermal Cyclers/C1000 Touch Thermal Cycler ->MTS::LGTW9415##Life Science Research/Products/Amplification - PCR/PX1 PCR Plate Sealer ->MTS::M9ZR3T15## Life Science Research/Solutions/Technologies/PCR ->MTS::LUSNYI15##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR ->MTS::LUSO4W8UU## Karen Moss What 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 AM 11/28/22 12:28 AM AE,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 en LSR /LSR/Technologies/Digital_PCR N 0 Introduction to Digital PCR /en-us/applications-technologies/applications-technologies/droplet-digital-pcr-ddpcr-technology?ID=MDV300E8Z

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.

This section provides an overview of droplet digital PCR technology, how ddPCR works, and what the advantages and benefits of ddPCR are.

Related topics: Planning Droplet Digital PCR Experiments, Absolute Quantification of PCR Targets with the Droplet Digital™ PCR System

Our PrimePCR™ Assays for Droplet Digital™ PCR are designed and validated for copy number variation, rare mutation detection, NGS library quantification, and high-sensitivity gene expression studies.

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What is Droplet Digital PCR?

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.

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.

Sample partitioning is the key to Droplet Digital PCR

Sample partitioning is the key to droplet digital PCR. 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.

 

How Does Droplet Digital PCR Work?

ddPCR technology uses a combination of microfluidics and proprietary surfactant chemistries to divide PCR samples into water-in-oil droplets (Hindson et al. 2011). 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 Poisson statistics to determine the target DNA template concentration in the original sample.

 

Advantages of ddPCR over Other Digital PCR Technologies

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.

 

The Benefits of Droplet Digital PCR

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.

The benefits of ddPCR technology include:

  • Absolute quantification — 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
  • Unparalleled precision — the massive sample partitioning afforded by ddPCR enables the reliable measurement of small fold differences in target DNA sequence copy numbers among samples
  • Increased signal-to-noise ratio — 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
  • Removal of PCR bias — error rates are reduced by removing the amplification efficiency reliance of qPCR, enabling the detection of small (1.2-fold) differences
  • Simplified quantification — neither calibration standards nor a reference (the ΔΔCq method) is required for absolute quantification
  • Reduced consumable costs — reaction volumes are in the pico- to nanoliter ranges, reducing reagent use and the sample quantity required for each data point
  • Lower equipment costs — the emulsion-based reaction system means that the PCR reactions can be performed in a standard thermal cycler without complex chips or microfluidics
  • Superior partitioning — 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
 

QX200™ Droplet Digital™ PCR System

Bio-Rad’s QX200 Droplet Digital PCR (ddPCR) System 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.

The ddPCR System can be used to:

  • Detect rare DNA target copies with unmatched sensitivity
  • Determine copy number variation with unrivaled accuracy
  • Measure gene expression levels with exquisite precision

QX100 Droplet Digital PCR system

QX200 Droplet Digital PCR System. QX200 Droplet Reader (left) and the QX200 Droplet Generator (right).

 

QX200 Droplet Digital PCR System Workflow

This 3-D animated video describes the principles and process of ddPCR using Bio-Rad's Droplet Digital PCR System.

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.

Step 0: Prepare PCR-Ready Samples prior to Starting ddPCR

Step 0: Prepare PCR-Ready Samples prior to Starting ddPCR

Step 1: Droplet Generation

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.

Step 1: Droplet Generation

Step 2: PCR Amplification of Droplets

Droplets are transferred to a 96-well plate for PCR amplification in any compatible thermal cycler.

Step 2: PCR Amplification of Droplets

Step 3: Droplet Reading

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.

Step 3: Droplet Reading

Digital droplet reading

 


Digital droplet reading. Fluorescence measurements for each droplet in two optical channels
are used to count the numbers of positive and negative droplets per sample.


 


Step 4: Analyze Results

Step 4: Analyze Results

Positive droplets, containing at least one copy of the target, exhibit increased fluorescence over negative droplets. 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.

Sample results from a ddPCR experiment

Sample results from a ddPCR experiment. 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.

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:

  • FAM-negative, HEX-negative
  • FAM-positive, HEX-negative
  • FAM-negative, HEX-positive
  • FAM-positive, HEX-positive

Mutation Detection

2-D plot of droplet fluorescence.


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.

Concentration results

Concentration results. Sample concentrations are plotted as copies per µl.

 

Step 5: Visualize Data

digital-pcr-visualize-data-step5

 

References

 

Related Content

 
Literature
Number Description Download
6237 QX100&#153; Droplet Digital&#153; PCR System Brochure, Rev C Click to download
6311 QX200™ Droplet Digital™ PCR System Brochure, Rev C Click to download
6407 Droplet Digital PCR Applications Guide Click to download
6554 Droplet Digital&#8482; PCR: Detection of DNA Methylation Application Note, Rev A Click to download
6277 Probing Copy Number Variations Using Bio-Rad's QX100 Droplet Digital PCR System, Rev A Click to download
6451 Droplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital PCR System Application Note, Rev A Click to download
6578 Droplet Digital&trade; PCR: Multiplex Detection of <em>KRAS</em> Mutations in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples Application Note, Rev A Click to download
6679 Droplet Digital&trade; PCR: Multiplex Screening of <em>KRAS</em> Mutations in Cell-Free DNA Colorectal Cancer Samples Application Note, Rev A Click to download
 
 
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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&trade; PCR System</a></p> <div class="bannerAT" style="margin-left:10px;"> <div class="bannerText ddpcrText"> <h2 class="banner_header">Validated ddPCR&trade; Assays</h2> <p>Our PrimePCR&trade; Assays for Droplet Digital&trade; PCR are designed and validated for copy number variation, rare mutation detection, NGS library quantification, and high-sensitivity gene expression studies.</p> <a class="linkgeneration" href="/_locale/product/digital-pcr/digital-pcr-assays?at_banner=Droplet Digital PCR ddPCR Technology&amp;at_banner_rev=Droplet Digital PCR ddPCR Technology&amp;at_banner_e=c">Learn more &raquo;</a></div> <img src="/webroot/web/images/lsr/global/english/solutions/lab-validated-ddpcr-assays.jpg" alt="Lab-Validated ddPCR&trade; Assays" width="615" height="120" /></div> <script src="/webroot/web/js/countrySpecific-min.js" type="text/javascript"></script> <script type="text/javascript"><!-- $(document).ready(function(){ setSterlingUrlsToHtmlHrefVariables(); $('a.linkgeneration').each(function(){ $(this).attr('href', $(this).attr('href').replace('_locale', languageCode + '-' + countryCode).replace('_verticalUrl', currentVerticalUrlTitle).replace('_defaultVerticalUrl', defaultVerticalUrlTitle).replace('_feedbackCMSID',feedbackCMSID)); }); }); // --></script> 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> <div class="top"><a href="#helptop">Back to Top</a></div> 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> <div class="top"><a href="#helptop">Back to Top</a></div> 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> <div class="top"><a href="#helptop">Back to Top</a></div> 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> &mdash; 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> &mdash; 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> &mdash; 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 &plusmn;10% precision in quantification</li> <li><strong>Removal of PCR bias</strong> &mdash; 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> &mdash; neither calibration standards nor a reference (the <em>&Delta;&Delta;Cq</em> method) is required for absolute quantification</li> <li><strong>Reduced consumable costs</strong> &mdash; 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> &mdash; 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>&mdash; ddPCR technology yields 20,000 droplets per 20 &micro;l 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> <div class="top"><a href="#helptop">Back to Top</a></div> QX200&trade; Droplet Digital&trade; PCR System <p>Bio-Rad&rsquo;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 &micro;l 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&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;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&trade; 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">&nbsp;</p> <p class="caption"><strong><br /></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><br /></strong></p> <p class="caption">&nbsp;</p> </td> </tr> </tbody> </table> <p><strong><br /></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/&micro;l.</p> <strong> <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> </strong> <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/&micro;l input sample and then reports the target DNA concentration in the form of copies per &micro;l 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 &micro;l.</p> <p class="caption">&nbsp;</p> <strong> <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> </strong> <div class="top"><a href="#helptop">Back to Top</a></div> References <ul> <li>Bizouarn F <a href="http://www.genengnews.com/gen-articles/digital-pcr-improving-nucleic-acid-quantification/4093/" target="_blank">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&ndash;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&ndash;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&ndash;449. PMID: 1389177</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> 6450 /templatedata/internet/documentation/data/LSR/Literature/6450.xml 6237 /templatedata/internet/documentation/data/LSR/Literature/6237.xml 6311 /templatedata/internet/documentation/data/LSR/Literature/6311.xml 6407 /templatedata/internet/documentation/data/LSR/Literature/6407_1393524927.xml 6554 /templatedata/internet/documentation/data/LSR/Literature/6554_1399586432.xml 6277 /templatedata/internet/documentation/data/LSR/Literature/6277.xml 6451 /templatedata/internet/documentation/data/LSR/Literature/6451_1387577658.xml 6578 /templatedata/internet/documentation/data/LSR/Literature/6578_1411685676.xml 6679 /templatedata/internet/documentation/data/LSR/Literature/6679_1418173918.xml Life Science Research/Products/Amplification - PCR/Digital PCR/QX100 Droplet Digital PCR ->MTS::M9HE3XE8Z##Life Science Research/Products/Amplification - PCR/Thermal Cyclers/C1000 Touch Thermal Cycler ->MTS::LGTW9415##Life Science Research/Products/Amplification - PCR/PX1 PCR Plate Sealer ->MTS::M9ZR3T15## Life Science Research/Solutions/Technologies/PCR ->MTS::LUSNYI15##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR ->MTS::LUSO4W8UU## Karen Moss What 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 AM 11/28/22 12:28 AM AE,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 en LSR /LSR/Technologies/Digital_PCR N 0
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