Name: pcr cycle for dna amplification
Uploaded: 2026-08-26T08:19:52.163Z
Duration: 2 min 7 s
Channel: JoVE

Das Ziel der PCR (Polymerase-Kettenreaktion) ist die Amplifikation einer genetischen Sequenz.
 In diesem Beispiel wird ein interessierendes Gen aus gereinigter DNA amplifiziert.
 Um die Amplifikation durchzuf&#xFC;hren, ist eine Polymerase erforderlich, um die neue DNA zu synthetisieren.
 Ein Primer, ein kurzes St&#xFC;ck einzelstr&#xE4;ngiger DNA, das Homologie zu dem interessierenden Gen aufweist, wird ebenfalls ben&#xF6;tigt.
 Schlie&#xDF;lich werden einzelne Nukleotide, sogenannte Desoxynukleotidtriphosphate oder dNTPs, verwendet, um den neuen Strang herzustellen.
 Der erste Schritt bei der PCR besteht darin, die Mischung zu erhitzen, wodurch die DNA denaturiert wird.
 Als n&#xE4;chstes wird die Reaktion abgek&#xFC;hlt und die Primer werden an ihren homologen Bereich angelagert.
 Einmal gebunden, wird die Reaktion auf eine f&#xFC;r die Polymerase optimale Temperatur erhitzt.
 Die Polymerase erkennt dann den Primer-DNA-Komplex und beginnt die Synthese des neuen Strangs unter Verwendung der dNTPs in der L&#xF6;sung.
 Die Reaktion wird dann erhitzt und verl&#xE4;uft &#xFC;ber weitere Zyklen wie beschrieben.
 Nach dem dritten Zyklus gibt es insgesamt acht Kopien des Gens.
 Nach dem vierten Zyklus 16 Exemplare, und dem f&#xFC;nften Zyklus, 32 Exemplare.
 Die Anzahl der Kopien wird weiterhin exponentiell zunehmen.
 Nach 30 Zyklen gibt es etwas mehr als 1 Milliarde Exemplare.

Garibyan, Lilit und Nidhi Avashia. "Forschungstechniken leicht gemacht: Polymerase-Kettenreaktion (PCR)." The Journal of Investigative Dermatology 133, Nr. 3 (März 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Quelltext</a>]

Pray, Leslie A. "Die biotechnologische Revolution: PCR und die Verwendung von reverser Transkriptase zum Klonen exprimierter Gene." Nature Education 1, Nr. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Quelle</a>]

<h4>&#xDC;berblick</h4>
 
 Die Polymerase-Kettenreaktion (englisch polymerase chain reaction, PCR) ist eine weit verbreitete Methode f&#xFC;r das Kopieren von DNA-Abschnitten. Aufgrund der exponentiellen Amplifikation kann die PCR innerhalb weniger Stunden Millionen oder Milliarden von DNA-Kopien herstellen. In einer PCR-Reaktion amplifiziert ein hitzebest&#xE4;ndiges DNA-Polymerase-Enzym die urspr&#xFC;ngliche DNA mit Hilfe einer Reihe von Temperatur&#xE4;nderungen in dem Thermocycler.
 
 <h4>Die PCR ist eine vielseitige Methode, die die Molekularbiologie revolutionierte</h4>
 
 Kary Mullis entwickelte die PCR im Jahr 1983. Daf&#xFC;r erhielt er im Jahr 1993 den Nobelpreis f&#xFC;r Chemie. Als relativ schnelle, kosteng&#xFC;nstige und pr&#xE4;zise Methode f&#xFC;r das Kopieren einer DNA-Sequenz wurde PCR zu einem unsch&#xE4;tzbaren Werkzeug f&#xFC;r zahlreiche Anwendungen. Dazu geh&#xF6;ren beispielsweise das molekulare Klonen, die Mutagenese, der Nachweis von Krankheitserregern, die Analyse der Genexpression, die Quantifizierung und Sequenzierung von DNA sowie die Diagnose genetischer Krankheiten.
 
 <h4>Der PCR-Reaktionszyklus</h4>
 
 PCR ahmt den nat&#xFC;rlichen DNA-Replikationsprozess nach, der in Zellen abl&#xE4;uft. Das Reaktionsgemisch enth&#xE4;lt eine zu kopierende Original-DNA-Sequenz (Template), ein Paar kurze DNA-Molek&#xFC;le (die Primer genannt werden), freie DNA-Bausteine, die Desoxynukleotidtriphosphate (die dNTPs), und ein spezialisiertes DNA-Polymerase-Enzym.
 
 PCR umfasst eine Reihe von Schritten bei hohen Temperaturen. Daher wird ein DNA-Polymerase-Enzym gebraucht, das bei solchen Temperaturen aktiv ist. Die am h&#xE4;ufigsten verwendete DNA-Polymerase ist die Taq-Polymerase, benannt nach Thermus aquaticus, dem Bakterium, aus dem die Polymerase isoliert wurde. Die DNA-Polymerase ist nicht dazu in der Lage, ein DNA-Molek&#xFC;l von Grund auf oder neu zu synthetisieren (de novo). Stattdessen f&#xFC;gt die DNA-Polymerase an die kurzen DNA-Molek&#xFC;le, die Primer, an. Diese binden sich durch komplement&#xE4;re Basenpaarung an die Original-DNA. Die Primer haben eine freie 3&#x2019;-Hydroxylgruppe, an welche die DNA-Polymerase neue dNTPs binden kann. Es gibt vier Arten von dNTPs in einer PCR-Reaktion, eines f&#xFC;r jedes Nukleotid im DNA-Molek&#xFC;l: dATP, dCTP, dGTP und dTTP.
 
 Der PCR-Zyklus besteht aus drei Schritten: Denaturierung, Annealing und DNA-Synthese.
 
 <ol>
 <li>Denaturierung. Der PCR-Zyklus beginnt mit dem Erhitzen des Reaktionsgemisches auf eine hohe Temperatur. Dabei wird die DNA-Doppelhelix in zwei Str&#xE4;nge aufgetrennt. Dieser &#x201C;Schmelz&#x201D;-Prozess findet normalerweise bei Temperaturen zwischen 90&#xB0;C&#x2013;100&#xB0;C statt.</li>
 <li>Annealing. Das Reaktionsgemisch wird schnell abgek&#xFC;hlt (normalerweise auf 50&#xB0;C&#x2013;65&#xB0;C, wodurch die beiden Primer an ihre komplement&#xE4;ren Sequenzen auf den Template-DNA-Str&#xE4;ngen binden k&#xF6;nnen.</li>
 <li>DNA-Synthese (Primer-Verl&#xE4;ngerung). Das Reaktionsgemisch wird erneut erhitzt, diesmal auf eine Temperatur (normalerweise 60&#xB0;C&#x2013;75&#xB0;C, die es der DNA-Polymerase erlaubt, die Primer durch das Hinzuf&#xFC;gen von dNTPs, die sich mit den Basen der Template-DNA paaren, zu verl&#xE4;ngern.</li>
 </ol>
 
 Eine typische PCR beinhaltet 20-40 wiederholte Zyklen dieser drei Schritte, die im Thermocycler stattfinden. Da sich die Anzahl der DNA-Molek&#xFC;le in jedem Zyklus verdoppelt, wird die DNA exponentiell vervielf&#xE4;ltigt.
 
 <h4>Grenzen der PCR-Methode</h4>
 
 Wenn ein Wissenschaftler einen bestimmten Abschnitt des Genoms amplifizieren will, muss er zumindest einen Teil der gew&#xFC;nschten DNA-Sequenz kennen, um einen geeigneten Primer zu entwerfen. Ein weiteres potenzielles Problem ist die unspezifische Annealierung von Primern an teilweise &#xE4;hnliche DNA-Sequenzen, welches zur Amplifikation von nicht gew&#xFC;nschten DNA-Sequenzen f&#xFC;hrt. Dieses Problem l&#xE4;sst sich durch die Optimierung der Reaktionsbedingungen kontrollieren. Da die PCR eine hochempfindliche Detektionsmethode ist, ist sie auch f&#xFC;r Kontaminationen anf&#xE4;llig. Selbst Spuren kontaminierender DNA k&#xF6;nnen zu irref&#xFC;hrenden Ergebnissen f&#xFC;hren. Zus&#xE4;tzlich, k&#xF6;nnen die in der PCR verwendeten DNA-Polymerasen fehleranf&#xE4;llig sein. Kommt es innerhalb der ersten PCR-Zyklen zu einer Mutation, tr&#xE4;gt der gr&#xF6;&#xDF;te Teil der amplifizierten DNA diese Mutation.

Overview

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce ...

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pcr cycle for dna amplification

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler.

PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene expression analysis, DNA quantitation, sequencing, and genetic disease diagnosis.

PCR copies DNA in a way that resembles natural DNA replication. The reaction mixture includes template DNA, short DNA primers, free DNA building blocks called deoxynucleotide triphosphates, or dNTPs, and a special DNA polymerase. The most common enzyme is Taq polymerase, which comes from the bacterium Thermus aquaticus.

DNA polymerase cannot start a DNA strand from scratch. It needs primers, which are short DNA molecules that bind to the template by complementary base pairing. The primers provide a free 3' hydroxyl group so the polymerase can add new dNTPs. The four dNTPs used in PCR are dATP, dCTP, dGTP, and dTTP.

Each PCR cycle has three steps: denaturation, annealing, and DNA synthesis. During denaturation, the mixture is heated to about 90°C to 100°C, which separates the double helix into two strands. During annealing, the mixture cools to about 50°C to 65°C so the primers can bind to their matching sequences.

During DNA synthesis, also called primer extension, the mixture is heated again to about 60°C to 75°C. At this stage, DNA polymerase extends the primers by adding dNTPs that match the template strand. A typical PCR uses 20 to 40 cycles in the thermocycler, and the DNA amount doubles each cycle, so amplification is exponential.

PCR has limits that scientists must watch for. To copy a specific genome region, the target sequence must be known well enough to design the right primers. Primers can also bind to similar non-target DNA, which can create unwanted products, but reaction conditions can be adjusted to reduce this problem.

Because PCR is very sensitive, contamination can cause misleading results even when only trace amounts of outside DNA are present. The DNA polymerases used in PCR can also make mistakes. If a mutation happens early in the cycle, most of the amplified DNA will carry that mutation.

PCR Cycle for DNA Amplification

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Learn how PCR amplifies DNA through denaturation, annealing, and extension, plus the role of primers, Taq polymerase, and common PCR limits.

PCR Cycle for DNA Amplification | JoVE K12

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PCR Cycle for DNA Amplification

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