Changes

<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>

<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" />

<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>

<p>&nbsp;</p>

<h3><span class="mw-headline">Isolation of genomic DNA</span></h3>

<p>&nbsp;</p>

<h2><span class="mw-headline">References</span></h2>

<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>