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<li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/workshop-details.html"> Workshop Details </a></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/workshop-video.html"> Workshop Video </a></li><li class="has-children is-open"><div class="acnav__label"> <i class="arrow up"></i><a class="active" href="/ncbi/workshops/2023-09_Primer-BLAST/PCR.html"> The Polymerase Chain Reaction </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: block;"><li class="li nav-item"><a class="notactive nav-link link10314" href="/ncbi/workshops/2023-09_Primer-BLAST/PCR.html#PCR">Advent of PCR</a></li><li class="li nav-item"><a class="notactive nav-link link10325" href="/ncbi/workshops/2023-09_Primer-BLAST/PCR.html#Primers">Designing Primers</a></li></ul></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/PB_description.html"> What is Primer-BLAST? </a></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/PB_access.html"> Accessing Primer-BLAST </a></li><li class="has-children"><div class="acnav__label"> <i class="arrow down"></i><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html"> The Interface </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: none;"><li class="li nav-item"><a class="notactive nav-link link10639" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Template">Input Sequences</a></li><li class="li nav-item"><a class="notactive nav-link link106410" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Exon">Exon/Intron Selection</a></li><li class="li nav-item"><a class="notactive nav-link link106511" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Specificity">Specificity Check</a></li><li class="li nav-item"><a class="notactive nav-link link106612" href="/ncbi/workshops/2023-09_Primer-BLAST/interface.html#Advanced">Advanced Parameters</a></li></ul></li><li class="has-children"><div class="acnav__label"> <i class="arrow down"></i><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/results.html"> Results Page </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: none;"><li class="li nav-item"><a class="notactive nav-link link107714" href="/ncbi/workshops/2023-09_Primer-BLAST/results.html#Results">Results</a></li><li class="li nav-item"><a class="notactive nav-link link107815" href="/ncbi/workshops/2023-09_Primer-BLAST/results.html#Recent">Recent Results</a></li></ul></li><li class="has-children"><div class="acnav__label"> <i class="arrow down"></i><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html"> Practice Examples </a></div><ul class="acnav__list acnav__list--level2" style="list-style-type: none; display: none;"><li class="li nav-item"><a class="notactive nav-link link108917" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html#Ex1">Example 1: MPO mRNA</a></li><li class="li nav-item"><a class="notactive nav-link link1081018" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html#Ex2">Example 2: MPO Exon</a></li><li class="li nav-item"><a class="notactive nav-link link1081119" href="/ncbi/workshops/2023-09_Primer-BLAST/examples.html#Ex3">Example 3: Whale myoglobins</a></li></ul></li><li><a class="notactive" href="/ncbi/workshops/2023-09_Primer-BLAST/job_ids.html"> Today's Job IDs </a></li>
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<h1>The Polymerase Chain Reaction</h1>
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<h2><a id="PCR"></a>The Advent of PCR</h2>
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<p>In the mid-1980's Kary Mullis and others devised a method to rapidly amplify a targeted region of DNA into milliions of copies. The method used <em>E. coli </em>polymerase-mediated extension of DNA oligonucletides (primers) that were complementary to regions flanking the target<em>. </em>They named the method the polymerase chain reaction (PCR). Mullis received the 1993 Nobel Prize in Chemistry for inventing the technique. <br /><em><br /></em>Standard PCR involves 1) denaturing a double-stranded target DNA at high temperature, 2) lowering the temperature to allow the primers to bind (anneal), and 3) raising the temperature and extending the primers using a thermostable polymerase. Repeated cycles result in an exponential growth of the number of copies of the target DNA. </p>
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<p>It's remarkable that the first experiments used a manual process involving water baths and a polymerase that was unstable at high temperatures. The advent of thermostable polymerases from thermophilic bacteria and archaea combined with computer-controlled thermocycling machines have made PCR a standard laboratory technique that is used in a wide range of molecular biology procedures including measuring gene expression, amplifying targets for sequencing and cloning, and assaying sequence variation.</p>
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<br />
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<h3 style="padding-left: 40px;">PCR cycle: denaturation, annealing, extension</h3>
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<img src="/ncbi/workshops/2022-10_Primer-BLAST/Images/PCR.JPG" alt="Graphic depicting the cycles of the polymerase chain reaction" width="900" /><br /><br />
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<h3 style="padding-left: 40px;">Details of primer binding and extension</h3>
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<p><img src="/ncbi/workshops/2022-10_Primer-BLAST/Images/Primer_Binding_Extension.jpg" alt="Image of details of primer binding and extension in PCR" width="900" /><br /><br /></p>
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<h2><a id="Primers"></a>Designing primers for PCR</h2>
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<p>An important aspect of PCR technique is identifying appropriate primers that:</p>
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<ol>
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<li>Match the target sequence in the correct locations</li>
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<li>Have compatible thermodynamic properties with the template such as denaturation (melting) temperature (Tm)</li>
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<li>Don't have stable secondary structures, self-complementarity, or other competing binding problems</li>
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<li>Will not amplify unintended products from the DNA sample</li>
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</ol>
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<p>The first three items above are addressed by most primer designing software including the popular and free Primer3. The last item, specificity checking, requires searching the primers against a database of background DNA sequences to look for unintended matches. This is a job for BLAST.</p>
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