Polymerase Chain Reaction(PCR) - Revision history

J at 05:26, 9 March 2008

2008-03-09T05:26:28Z

J at 03:38, 22 February 2008

2008-02-22T03:38:14Z

J at 03:38, 22 February 2008

2008-02-22T03:38:03Z

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Yslee at 09:26, 2 February 2006

2006-02-02T09:26:47Z

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[[Image:PCR.gif]]<br /><br /><font color="#008000" size="2"><html><a href="http://www.science2discover.com">www.science2discover.com</a></html></font>

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 <p>Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme derived from the bacterium <em>Thermus aquaticus</em>. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These different temperature steps are necessary to bring about physical separation of the strands in a DNA double helix (DNA melting), and permit DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The power and selectivity of PCR are primarily due to selecting primers that are highly complementary to the DNA region targeted for amplification, and to the thermal cycling conditions used.</p>
 <p>Developed in 1983 by Kary Mullis,<sup class="reference" id="_ref-Bartlett_.26_Stirling_0">[1]</sup> PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensics and paternity testing); and the detection and diagnosis of infectious diseases. Mullis won the Nobel Prize for his work on PCR.<sup class="reference" id="_ref-Karry_Mullis_Nobel_Lecture_0">[2]</sup></p>
 <p>&nbsp;</p>
-<h2><span class="mw-headline">PCR principle and procedure</span></h2>
+<p><span class="mw-headline"><font size="5">PCR principle and procedure</font></span></p>
 <div class="thumb tright">
 <div class="thumbinner" style="WIDTH: 202px"><img class="thumbimage" height="194" alt="Figure 1a: An old thermal cycler for PCR" width="200" border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/d/da/Pcr_machine.jpg/200px-Pcr_machine.jpg" />
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 <p>In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.<sup class="reference" id="_ref-3">[10]</sup><sup class="reference" id="_ref-4">[11]</sup> Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR reactions from potential DNA contaminants.<sup class="reference" id="_ref-molecular_cloning_1">[4]</sup> This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.</p>
 <p>&nbsp;</p>
-<h2><span class="mw-headline">Application of PCR</span></h2>
+<p><span class="mw-headline"><font size="5">Application of PCR</font></span></p>
 <p>&nbsp;</p>
 <h3><span class="mw-headline">Isolation of genomic DNA</span></h3>
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 <p>&nbsp;</p>
 <h2><span class="mw-headline">References</span></h2>
-<div class="references-small" style="-moz-column-count: 2; -webkit-column-count: 2; column-count: 2">
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 <ol class="references">
      <li id="_note-Bartlett_.26_Stirling"><strong>^</strong> Bartlett &amp; Stirling (2003)&mdash;A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6 </li>

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 <p>Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme derived from the bacterium <em>Thermus aquaticus</em>. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These different temperature steps are necessary to bring about physical separation of the strands in a DNA double helix (DNA melting), and permit DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The power and selectivity of PCR are primarily due to selecting primers that are highly complementary to the DNA region targeted for amplification, and to the thermal cycling conditions used.</p>
 <p>Developed in 1983 by Kary Mullis,<sup class="reference" id="_ref-Bartlett_.26_Stirling_0">[1]</sup> PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensics and paternity testing); and the detection and diagnosis of infectious diseases. Mullis won the Nobel Prize for his work on PCR.<sup class="reference" id="_ref-Karry_Mullis_Nobel_Lecture_0">[2]</sup></p>
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 <p>&nbsp;</p>
 <h2><span class="mw-headline">PCR principle and procedure</span></h2>
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-<div class="thumbinner" style="WIDTH: 202px"><img class="thumbimage" height="194" alt="Figure 1a: An old thermal cycler for PCR" src="http://upload.wikimedia.org/wikipedia/commons/thumb/d/da/Pcr_machine.jpg/200px-Pcr_machine.jpg" width="200" border="0" />
+<div class="thumbinner" style="WIDTH: 202px"><img class="thumbimage" height="194" alt="Figure 1a: An old thermal cycler for PCR" width="200" border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/d/da/Pcr_machine.jpg/200px-Pcr_machine.jpg" />
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 <strong>Figure 1a</strong>: An old thermal cycler for PCR</div>
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-<div class="thumbinner" style="WIDTH: 182px"><img class="thumbimage" height="203" alt="Figure 1b: A very old three-temperature thermal cycler for PCR" src="http://upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Primitive_PCR_machine_for_scrap.JPG/180px-Primitive_PCR_machine_for_scrap.JPG" width="180" border="0" />
+<div class="thumbinner" style="WIDTH: 182px"><img class="thumbimage" height="203" alt="Figure 1b: A very old three-temperature thermal cycler for PCR" width="180" border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Primitive_PCR_machine_for_scrap.JPG/180px-Primitive_PCR_machine_for_scrap.JPG" />
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 <strong>Figure 1b</strong>: A very old three-temperature thermal cycler for PCR</div>
 </div>
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 <h3><span class="mw-headline">Procedure</span></h3>
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-<div class="thumbinner" style="WIDTH: 302px"><img class="thumbimage" height="675" alt="Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here." src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/87/PCR.svg/300px-PCR.svg.png" width="300" border="0" />
+<div class="thumbinner" style="WIDTH: 302px"><img class="thumbimage" height="675" alt="Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here." width="300" border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/87/PCR.svg/300px-PCR.svg.png" />
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-<div class="magnify"><img height="11" alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" width="15" /></div>
+<div class="magnify"><img height="11" alt="" width="15" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" /></div>
 <strong>Figure 2</strong>: Schematic drawing of the PCR cycle. <strong>(1) Denaturing at 94-96&deg;C. (2) Annealing at ~65&deg;C (3) Elongation at 72&deg;C</strong>. Four cycles are shown here.</div>
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-<div class="thumbinner" style="WIDTH: 302px"><img class="thumbimage" height="283" alt="Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs." src="http://upload.wikimedia.org/wikipedia/en/thumb/d/d0/Roland_Gel.JPG/300px-Roland_Gel.JPG" width="300" border="0" />
+<div class="thumbinner" style="WIDTH: 302px"><img class="thumbimage" height="283" alt="Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs." width="300" border="0" src="http://upload.wikimedia.org/wikipedia/en/thumb/d/d0/Roland_Gel.JPG/300px-Roland_Gel.JPG" />
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-<div class="magnify"><img height="11" alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" width="15" /></div>
+<div class="magnify"><img height="11" alt="" width="15" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" /></div>
 <strong>Figure 3</strong>: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.</div>
 </div>
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 <p>Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms.</p>
 <div class="thumb tright">
-<div class="thumbinner" style="WIDTH: 202px"><img class="thumbimage" height="301" alt="Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint." src="http://upload.wikimedia.org/wikipedia/commons/thumb/0/02/Pcr_fingerprint.png/200px-Pcr_fingerprint.png" width="200" border="0" />
+<div class="thumbinner" style="WIDTH: 202px"><img class="thumbimage" height="301" alt="Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint." width="200" border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/0/02/Pcr_fingerprint.png/200px-Pcr_fingerprint.png" />
 <div class="thumbcaption">
-<div class="magnify"><img height="11" alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" width="15" /></div>
+<div class="magnify"><img height="11" alt="" width="15" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" /></div>
 <strong>Figure 4</strong>: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.</div>
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-     <li><a class="external text" title="http://www.sciam.com/article.cfm?articleID=00035C6C-229B-1C74-9B81809EC588EF21" href="http://www.sciam.com/article.cfm?articleID=00035C6C-229B-1C74-9B81809EC588EF21" rel="nofollow">PCR at Home</a> - Amateur Scientist article in the July 2000 issue of <a title="Scientific American" href="http://en.wikipedia.org/wiki/Scientific_American">Scientific American</a> on performing PCR reactions with low-cost household materials. </li>
+     <li><a class="external text" title="http://www.sciam.com/article.cfm?articleID=00035C6C-229B-1C74-9B81809EC588EF21" rel="nofollow" href="http://www.sciam.com/article.cfm?articleID=00035C6C-229B-1C74-9B81809EC588EF21">PCR at Home</a> - Amateur Scientist article in the July 2000 issue of <a title="Scientific American" href="http://en.wikipedia.org/wiki/Scientific_American">Scientific American</a> on performing PCR reactions with low-cost household materials. </li>
-     <li><a class="external text" title="http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&amp;Sect2=HITOFF&amp;p=1&amp;u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&amp;r=1&amp;f=G&amp;l=50&amp;d=PALL&amp;RefSrch=yes&amp;Query=PN%2F4683202" href="http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&amp;Sect2=HITOFF&amp;p=1&amp;u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&amp;r=1&amp;f=G&amp;l=50&amp;d=PALL&amp;RefSrch=yes&amp;Query=PN%2F4683202" rel="nofollow">US Patent for PCR</a> </li>
+     <li><a class="external text" title="http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&amp;Sect2=HITOFF&amp;p=1&amp;u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&amp;r=1&amp;f=G&amp;l=50&amp;d=PALL&amp;RefSrch=yes&amp;Query=PN%2F4683202" rel="nofollow" href="http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&amp;Sect2=HITOFF&amp;p=1&amp;u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&amp;r=1&amp;f=G&amp;l=50&amp;d=PALL&amp;RefSrch=yes&amp;Query=PN%2F4683202">US Patent for PCR</a> </li>
-     <li><a class="external text" title="http://www.sumanasinc.com/webcontent/animations/content/pcr.html" href="http://www.sumanasinc.com/webcontent/animations/content/pcr.html" rel="nofollow">Narrated animation and step-through animation of PCR</a> - From the educational multimedia company Sumanas. Adobe Flash required. </li>
+     <li><a class="external text" title="http://www.sumanasinc.com/webcontent/animations/content/pcr.html" rel="nofollow" href="http://www.sumanasinc.com/webcontent/animations/content/pcr.html">Narrated animation and step-through animation of PCR</a> - From the educational multimedia company Sumanas. Adobe Flash required. </li>
-     <li><a class="external text" title="http://www.dnalc.org/ddnalc/resources/pcr.html" href="http://www.dnalc.org/ddnalc/resources/pcr.html" rel="nofollow">Step-through animation of PCR</a> - From <a title="Cold Spring Harbor Laboratory" href="http://en.wikipedia.org/wiki/Cold_Spring_Harbor_Laboratory">Cold Spring Harbor</a>'s Dolan DNA Learning Center. Adobe Flash required. </li>
+     <li><a class="external text" title="http://www.dnalc.org/ddnalc/resources/pcr.html" rel="nofollow" href="http://www.dnalc.org/ddnalc/resources/pcr.html">Step-through animation of PCR</a> - From <a title="Cold Spring Harbor Laboratory" href="http://en.wikipedia.org/wiki/Cold_Spring_Harbor_Laboratory">Cold Spring Harbor</a>'s Dolan DNA Learning Center. Adobe Flash required. </li>
 </ul>
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