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

- [Verteller] Het doel van PCR, polymerasekettingreactie, is het versterken van een genetische sequentie.
 In dit voorbeeld zal een gen van belang worden geamplificeerd uit gezuiverd DNA.
 Om de amplificatie uit te voeren, is een polymerase vereist om het nieuwe DNA te synthetiseren.
Een primer, een kort stuk enkelstrengs DNA die homologie deelt met het gen van belang, is ook nodig.
 Tenslotte worden enkele nucleotiden genaamd deoxynucleotide trifosfaten, of dNTP&#x27;s, gebruikt om de nieuwe streng te maken.
 De eerste stap in PCR is het verwarmen van het mengsel, die het DNA denatureert.
Vervolgens wordt de reactie afgekoeld en zullen de primers aan hun homoloog gebied gloeien.
 Eenmaal gebonden, wordt de reactie verhit tot een optimale temperatuur voor het polymerase.
 Het polymerase herkent vervolgens het primer-DNA-complex en begint de synthese van de nieuwe streng, met behulp van de dNTP&#x27;s in de oplossing.
De reactie wordt vervolgens verhit en verloopt zoals beschreven in de aanvullende cycli.
 Na de derde cyclus zijn er in totaal acht exemplaren van het gen.
 Na de vierde cyclus, zestien exemplaren.
 En na de vijfde cyclus, twee&#xEB;ndertig exemplaren.
 Het aantal exemplaren blijft exponentieel groeien.
 Na dertig cycli zijn er iets meer dan een miljard exemplaren.

<h4>Overzicht</h4>

De polymerasekettingreactie, of PCR, is een veelgebruikte techniek voor het kopi&#xEB;ren van DNA-segmenten. Dankzij exponenti&#xEB;le amplificatie kan PCR binnen slechts enkele uren miljoenen of miljarden DNA-kopie&#xEB;n produceren. Bij een PCR-reactie amplificeert een hittebestendig DNA-polymerase-enzym het oorspronkelijke DNA door een reeks temperatuurveranderingen in een geautomatiseerde machine die een thermocycler wordt genoemd.

<h4>PCR is een veelzijdige methode die een revolutie teweeg heeft gebracht in de moleculaire biologie</h4>

Kary Mullis ontwikkelde PCR in 1983, waarvoor hij in 1993 de Nobelprijs voor de Scheikunde ontving. Omdat het een relatief snelle, goedkope en nauwkeurige manier is om een DNA-sequentie te kopi&#xEB;ren, werd PCR een hulpmiddel van onschatbare waarde voor talloze toepassingen, waaronder moleculair klonen, genmutagenese, detectie van pathogenen, analyse van genexpressie, DNA-kwantificering en -sequencing, en diagnose van genetische ziekten.

<h4>De PCR-reactiecyclus</h4>

PCR bootst het natuurlijke DNA-replicatieproces na dat in cellen plaatsvindt. Het reactiemengsel omvat een sjabloon-DNA-sequentie die moet worden gekopieerd, een paar korte DNA-moleculen die primers worden genoemd, vrije DNA-bouwstenen die deoxynucleotide-trifosfaten (dNTP&#x27;s) worden genoemd, en een gespecialiseerd DNA-polymerase-enzym.

PCR omvat een reeks stappen bij hoge temperaturen, waarvoor een DNA-polymerase-enzym nodig is dat bij dergelijke temperaturen functioneel is. Het meest gebruikte DNA-polymerase is Taq-polymerase, genoemd naar Thermus Aquaticus, de bacterie waaruit het polymerase aanvankelijk werd ge&#xEF;soleerd. DNA-polymerase is niet in staat een DNA-molecuul helemaal opnieuw te synthetiseren, of de novo. In plaats daarvan voegt DNA-polymerase zich toe aan korte DNA-moleculen, primers genoemd, die via complementaire basenparing aan de DNA-sjabloon binden. De primers bieden een gratis 3&#x2019; hydroxylgroep waaraan DNA-polymerase nieuwe dNTP&#x27;s kan hechten. Er zijn vier soorten dNTP&#x27;s in een PCR, &#xE9;&#xE9;n voor elk nucleotide in het DNA-molecuul: dATP, dCTP, dGTP en dTTP.

Elke PCR-cyclus bestaat uit drie stappen: denaturatie, gloeien en DNA-synthese.

<ol>
	<li>Denaturatie. De PCR-cyclus wordt gestart door het reactiemengsel tot een hoge temperatuur te verwarmen, waardoor de dubbele DNA-helix in twee strengen wordt gescheiden. Dit &#x2018;smelten&#x2019; proces vindt meestal plaats bij een temperatuur van 90&#xB0;C&#x2013;100&#xB0;C.</li>
	<li>Gloeien. Het reactiemengsel wordt snel afgekoeld (meestal tot 50&#xB0;C-65&#xB0;C), waardoor de twee primers kunnen binden aan hun complementaire sequenties op de template-DNA-strengen.</li>
	<li>DNA-synthese (primerextensie). Het reactiemengsel wordt opnieuw verwarmd, deze keer tot een temperatuur (meestal 60&#xB0;C &#x2013; 75&#xB0;C) waarbij DNA-polymerase de primers kan verlengen door dNTP&#x27;s toe te voegen die paren met de basen in de matrijsstreng.</li>
</ol>



Een typische PCR omvat 20-40 herhaalde cycli van deze drie stappen, die plaatsvinden in de thermocycler. Omdat het aantal DNA-moleculen in elke cyclus wordt verdubbeld, wordt het DNA exponentieel geamplificeerd.

<h4>Beperkingen van PCR</h4>

Als de wetenschapper een specifiek stuk van het genoom wil amplificeren, moet de wetenschapper ten minste een deel van de doel-DNA-sequentie kennen om geschikte primers te kunnen ontwerpen. Een ander potentieel probleem is de niet-specifieke koppeling van primers aan gedeeltelijk vergelijkbare DNA-sequenties, wat leidt tot amplificatie van niet-doel-DNA. Dit probleem kan worden beheerst door de reactieomstandigheden te optimaliseren. Omdat het een zeer gevoelige detectiemethode is, is PCR ook kwetsbaar voor besmetting, en zelfs sporen van verontreinigend DNA kunnen misleidende resultaten veroorzaken. De DNA-polymerasen die bij PCR worden gebruikt, kunnen gevoelig zijn voor fouten. Als er binnen de eerste paar cycli een mutatie plaatsvindt, zal het grootste deel van het geamplificeerde DNA de mutatie dragen.

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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key terms and concepts

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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