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Digital PCR

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Digital Polymerase Chain Reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants and point mutations - and it is routinely used for clonal amplification of samples for "next-generation sequencing."

~~<table id="toc" class="toc tochidden">~~ ~~<tbody>~~ ~~<tr>~~ ~~<td>~~ ~~<div id="toctitle">~~ ~~<h2>Contents</h2>~~ ~~[show]</div>~~ ~~<ul style="display: none">~~ ~~<li class="toclevel-1 tocsection-1">1 PCR Basics</li>~~ ~~<li class="toclevel-1 tocsection-2">2 dPCR Working Principle</li>~~ ~~<li class="toclevel-1 tocsection-3">3 Development</li>~~ ~~<li class="toclevel-1 tocsection-4">4 References</li>~~ ~~<li class="toclevel-1 tocsection-5"~~> ~~</li>~~ ~~</ul>~~ ~~</td>~~ ~~</tr>~~ ~~</tbody></table>~~

<h2>PCR Basics</h2>

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase. Conventional PCR is based on the theory that amplification is exponential. Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample. However, many factors complicate this calculation, creating uncertainties and inaccuracies. These factors include the following: initial amplification cycles may not be exponential; PCR amplification eventually plateaus after an uncertain number of cycles; and low initial concentrations of target nucleic acid molecules may not amplify to detectable levels. However, the most significant limitation of PCR is that PCR amplification efficiency in a sample of interest may be different from that of reference samples. Since PCR is an exponential process, only twofold differences in amplification can be observed, greatly impacting the validity and precision of the results.

<li id="cite_note-0"><a href="#cite_ref-0">^</a> Sykes, PJ; Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA (1992). "Quantitation of targets for PCR by use of limiting dilution". Biotechniques 13 (3): 444–9. <a class="mw-redirect" title="PubMed Identifier" href="/wiki/PubMed_Identifier">PMID</a> <a class="external text" rel="nofollow" href="http://www.ncbi.nlm.nih.gov/pubmed/1389177~~" rel="nofollow~~">1389177</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Quantitation+of+targets+for+PCR+by+use+of+limiting+dilution&rft.jtitle=Biotechniques&rft.aulast=Sykes&rft.aufirst=PJ&rft.au=Sykes%2C%26%2332%3BPJ&rft.date=1992&rft.volume=13&rft.issue=3&rft.pages=444%E2%80%939&rft_id=info:pmid/1389177&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-1"><a href="#cite_ref-1">^</a> <a class="external text" rel="nofollow" href="http://www.cytonix.com/~~" rel="nofollow~~">"Cytonix"</a>. <a class="external free" rel="nofollow" href="http://www.cytonix.com/~~" rel="nofollow~~">http://www.cytonix.com/</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=bookitem&rft.btitle=Cytonix&rft.atitle=&rft_id=http%3A%2F%2Fwww.cytonix.com%2F&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-2"><a href="#cite_ref-2">^</a> Kalinina, O; Brown J, Silver J (1997). <a class="external text" rel="nofollow" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=146692~~" rel="nofollow~~">"Nanoliter scale PCR with TaqMan detection"</a>. Nucleic Acids Research 25 (10): 1999–2004. <a title="Digital object identifier" href="/wiki/Digital_object_identifier">doi</a>:<a class="external text" rel="nofollow" href="http://dx.doi.org/10.1093%2Fnar%2F25.10.1999~~" rel="nofollow~~">10.1093/nar/25.10.1999</a>. <a class="mw-redirect" title="PubMed Identifier" href="/wiki/PubMed_Identifier">PMID</a> <a class="external text" rel="nofollow" href="http://www.ncbi.nlm.nih.gov/pubmed/9115368~~" rel="nofollow~~">9115368</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Nanoliter+scale+PCR+with+TaqMan+detection&rft.jtitle=Nucleic+Acids+Research&rft.aulast=Kalinina&rft.aufirst=O&rft.au=Kalinina%2C%26%2332%3BO&rft.date=1997&rft.volume=25&rft.issue=10&rft.pages=1999%E2%80%932004&rft_id=info:doi/10.1093%2Fnar%2F25.10.1999&rft_id=info:pmid/9115368&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-3"><a href="#cite_ref-3">^</a> <a class="external text" rel="nofollow" href="http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=6,143,496.PN.&OS=PN/6,143,496&RS=PN/6,143,496~~" rel="nofollow~~">"U. S. Patent 6,143,496"</a>. <a class="external free" rel="nofollow" href="http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=6,143,496.PN.&OS=PN/6,143,496&RS=PN/6,143,496~~" rel="nofollow~~">http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=6,143,496.PN.&OS=PN/6,143,496&RS=PN/6,143,496</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=bookitem&rft.btitle=U.+S.+Patent+6%2C143%2C496&rft.atitle=&rft_id=http%3A%2F%2Fpatft.uspto.gov%2Fnetacgi%2Fnph-Parser%3FSect1%3DPTO1%26Sect2%3DHITOFF%26d%3DPALL%26p%3D1%26u%3D%252Fnetahtml%252FPTO%252Fsrchnum.htm%26r%3D1%26f%3DG%26l%3D50%26s1%3D6%2C143%2C496.PN.%26OS%3DPN%2F6%2C143%2C496%26RS%3DPN%2F6%2C143%2C496&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-4"><a href="#cite_ref-4">^</a> Vogelstein, B; Kinzler KW (1999). <a class="external text" rel="nofollow" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=17763~~" rel="nofollow~~">"Digital PCR"</a>. Proc Natl Acad Sci U S A. 96 (16): 9236–41. <a title="Digital object identifier" href="/wiki/Digital_object_identifier">doi</a>:<a class="external text" rel="nofollow" href="http://dx.doi.org/10.1073%2Fpnas.96.16.9236~~" rel="nofollow~~">10.1073/pnas.96.16.9236</a>. <a class="mw-redirect" title="PubMed Identifier" href="/wiki/PubMed_Identifier">PMID</a> <a class="external text" rel="nofollow" href="http://www.ncbi.nlm.nih.gov/pubmed/10430926~~" rel="nofollow~~">10430926</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Digital+PCR&rft.jtitle=Proc+Natl+Acad+Sci+U+S+A.&rft.aulast=Vogelstein&rft.aufirst=B&rft.au=Vogelstein%2C%26%2332%3BB&rft.date=1999&rft.volume=96&rft.issue=16&rft.pages=9236%E2%80%9341&rft_id=info:doi/10.1073%2Fpnas.96.16.9236&rft_id=info:pmid/10430926&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-5"><a href="#cite_ref-5">^</a> Pohl, G; Shih, I-M (2004). "Principle and applications of digital PCR". Expert Rev Mol Diagn 4 (1): 41–7. <a title="Digital object identifier" href="/wiki/Digital_object_identifier">doi</a>:<a class="external text" rel="nofollow" href="http://dx.doi.org/10.1586%2F14737159.4.1.41~~" rel="nofollow~~">10.1586/14737159.4.1.41</a>. <a class="mw-redirect" title="PubMed Identifier" href="/wiki/PubMed_Identifier">PMID</a> <a class="external text" rel="nofollow" href="http://www.ncbi.nlm.nih.gov/pubmed/14711348~~" rel="nofollow~~">14711348</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Principle+and+applications+of+digital+PCR&rft.jtitle=Expert+Rev+Mol+Diagn&rft.aulast=Pohl&rft.aufirst=G&rft.au=Pohl%2C%26%2332%3BG&rft.date=2004&rft.volume=4&rft.issue=1&rft.pages=41%E2%80%937&rft_id=info:doi/10.1586%2F14737159.4.1.41&rft_id=info:pmid/14711348&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-6"><a href="#cite_ref-6">^</a> Dressman, D; Yan H, Traverso G, Kinzler KW, Vogelstein B (2003). <a class="external text" rel="nofollow" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=166396~~" rel="nofollow~~">"Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations"</a>. Proc Natl Acad Sci USA 100 (15): 8817–22. <a title="Digital object identifier" href="/wiki/Digital_object_identifier">doi</a>:<a class="external text" rel="nofollow" href="http://dx.doi.org/10.1073%2Fpnas.1133470100~~" rel="nofollow~~">10.1073/pnas.1133470100</a>. <a class="mw-redirect" title="PubMed Identifier" href="/wiki/PubMed_Identifier">PMID</a> <a class="external text" rel="nofollow" href="http://www.ncbi.nlm.nih.gov/pubmed/12857956~~" rel="nofollow~~">12857956</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Transforming+single+DNA+molecules+into+fluorescent+magnetic+particles+for+detection+and+enumeration+of+genetic+variations&rft.jtitle=Proc+Natl+Acad+Sci+USA&rft.aulast=Dressman&rft.aufirst=D&rft.au=Dressman%2C%26%2332%3BD&rft.date=2003&rft.volume=100&rft.issue=15&rft.pages=8817%E2%80%9322&rft_id=info:doi/10.1073%2Fpnas.1133470100&rft_id=info:pmid/12857956&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-7"><a href="#cite_ref-7">^</a> Mikeska, T; Candiloro IL, Dobrovic A (2010). "The implications of heterogeneous DNA methylation for the accurate quantification of methylation". Epigenomics 2: 561–73. <a title="Digital object identifier" href="/wiki/Digital_object_identifier">doi</a>:<a class="external text" rel="nofollow" href="http://dx.doi.org/10.2217%2Fepi.10.32~~" rel="nofollow~~">10.2217/epi.10.32</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=The+implications+of+heterogeneous+DNA+methylation+for+the+accurate+quantification+of+methylation&rft.jtitle=Epigenomics&rft.aulast=Mikeska&rft.aufirst=T&rft.au=Mikeska%2C%26%2332%3BT&rft.date=2010&rft.volume=2&rft.pages=561%E2%80%9373&rft_id=info:doi/10.2217%2Fepi.10.32&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-8"><a href="#cite_ref-8">^</a> <a class="external text" rel="nofollow" href="http://www.Fluidigm.com/~~" rel="nofollow~~">"Fluidigm"</a>. <a class="external free" rel="nofollow" href="http://www.Fluidigm.com/~~" rel="nofollow~~">http://www.Fluidigm.com/</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=bookitem&rft.btitle=Fluidigm&rft.atitle=&rft_id=http%3A%2F%2Fwww.Fluidigm.com%2F&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-9"><a href="#cite_ref-9">^</a> <a class="external text" rel="nofollow" href="http://www.lifetechnologies.com/~~" rel="nofollow~~">"Life Technologies"</a>. <a class="external free" rel="nofollow" href="http://www.lifetechnologies.com/~~" rel="nofollow~~">http://www.lifetechnologies.com/</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=bookitem&rft.btitle=Life+Technologies&rft.atitle=&rft_id=http%3A%2F%2Fwww.lifetechnologies.com%2F&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li> <li id="cite_note-10"><a href="#cite_ref-10">^</a> <a class="external text" rel="nofollow" href="http://www.QuantaLife.com/~~" rel="nofollow~~">"QuantaLife"</a>. <a class="external free" rel="nofollow" href="http://www.QuantaLife.com/~~" rel="nofollow~~">http://www.QuantaLife.com/</a>.<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=bookitem&rft.btitle=QuantaLife&rft.atitle=&rft_id=http%3A%2F%2Fwww.QuantaLife.com%2F&rfr_id=info:sid/en.wikipedia.org:Digital_polymerase_chain_reaction"> </li>

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<ul>

<li>Brenan C, Morrison T, High throughput, nanoliter quantitative PCR. Drug Discovery Today: Technologies. 2; 247-253. 2005.</li>

<li><a class="external text" rel="nofollow" href="http://www.nature.com/nmeth/journal/v4/n10/full/nmeth1007-869.html~~" rel="nofollow~~">Blow N., PCR's next frontier, Nature Methods 4, 869 - 875 (2007) doi:10.1038/nmeth1007-869</a></li>

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