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

Le but de la PCR,R&#xE9;action en cha&#xEE;ne de la polym&#xE9;raseest d&#x27;amplifier une s&#xE9;quence g&#xE9;n&#xE9;tique. 
Dans cet exemple,un g&#xE8;ne d&#x27;int&#xE9;r&#xEA;t sera amplifi&#xE9;&#xE0; partir d&#x27;ADN purifi&#xE9;. 
Pour effectuer l&#x27;amplification,une polym&#xE9;rase est n&#xE9;cessairepour synth&#xE9;tiser le nouvel ADN. 
Une amorce, un petit morceau d&#x27;ADN monocat&#xE9;nairequi partage une homologie avec le g&#xE8;ne d&#x27;int&#xE9;r&#xEA;t,est &#xE9;galement n&#xE9;cessaire. 
Enfin, les nucl&#xE9;otides simples,appel&#xE9;s triphosphates de d&#xE9;soxynucl&#xE9;otides ou dNTP,sont utilis&#xE9;s pour fabriquer le nouveau brin. 
La premi&#xE8;re &#xE9;tape de la PCRest de chauffer le m&#xE9;lange,qui d&#xE9;nature l&#x27;ADN. 
Ensuite, la r&#xE9;action est refroidie,et les amorces seront appareill&#xE9;es &#xE0; leur r&#xE9;gion homologue. 
Une fois li&#xE9;e, la r&#xE9;action est chauff&#xE9;e&#xE0; une temp&#xE9;rature optimale pour la polym&#xE9;rase. 
La polym&#xE9;rase reconna&#xEE;t alorsle complexe de l&#x27;amorce d&#x27;ADN,et commence la synth&#xE8;se du nouveau brin,en utilisant les dNTP de la solution. 
La r&#xE9;action est ensuite chauff&#xE9;eet proc&#xE8;de tel que c&#x27;est d&#xE9;crit,d&#x27;un cycle &#xE0; l&#x27;autre. 
Apr&#xE8;s le troisi&#xE8;me cycle,il y a 8 copies du g&#xE8;ne au total. 
Apr&#xE8;s le quatri&#xE8;me cycle, 16 copies. 
Et cinqui&#xE8;me cycle, 32 copies. 
Le nombre d&#x27;exemplaires continuera de cro&#xEE;trede fa&#xE7;on exponentielle. 
Apr&#xE8;s 30 cycles,il y a un peu plus d&#x27;un milliard d&#x27;exemplaires.

Garibyan, Lilit et Nidhi Avashia. « Techniques de recherche simplifiées : réaction en chaîne par polymérase (PCR). » The Journal of Investigative Dermatology 133, n° 3 (mars 2013) : e6. [<a href="https://doi.org/10.1038/jid.2013.1 » target="__blank">Source</a>]

Pray, Leslie A. « La révolution biotechnologique : PCR et l’utilisation de la transcriptase inverse pour cloner les gènes exprimés. » Nature Education 1, n° 1 (2008) : 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553 » target="__blank">Source</a>]

<h4>Aper&#xE7;u</h4>
 
 La r&#xE9;action en cha&#xEE;ne par polym&#xE9;rase, ou PCR, est une technique largement utilis&#xE9;e pour copier des segments d&#x2019;ADN. En raison de l&#x2019;amplification exponentielle, la PCR peut produire des millions ou des milliards de copies d&#x2019;ADN en quelques heures. Dans une r&#xE9;action PCR, une enzyme d&#x2019;ADN polym&#xE9;rase r&#xE9;sistante &#xE0; la chaleur amplifie l&#x2019;ADN d&#x2019;origine par le biais d&#x2019;une s&#xE9;rie de changements de temp&#xE9;rature &#xE0; l&#x2019;int&#xE9;rieur d&#x2019;une machine automatis&#xE9;e appel&#xE9;e thermocycleur. 
 
 <h4>La PCR est une m&#xE9;thode polyvalente qui a r&#xE9;volutionn&#xE9; la biologie mol&#xE9;culaire</h4>
 
 Kary Mullis a mis au point la PCR en 1983, pour laquelle il a re&#xE7;u le prix Nobel de chimie en 1993. &#xC9;tant un moyen relativement rapide, peu co&#xFB;teux et pr&#xE9;cis de copier une s&#xE9;quence d&#x2019;ADN, la PCR est devenu un outil pr&#xE9;cieux pour de nombreuses applications, y compris le clonage mol&#xE9;culaire, la mutagen&#xE8;se des g&#xE8;nes, la d&#xE9;tection des agents pathog&#xE8;nes, l&#x2019;analyse de l&#x2019;expression g&#xE9;nique, la mesure de la quantit&#xE9; d&#x2019;ADN et le s&#xE9;quen&#xE7;age ainsi que le diagnostic des maladies g&#xE9;n&#xE9;tiques.
 
 <h4>Le cycle des r&#xE9;actions PCR</h4>
 
 La PCR imite le processus naturel de r&#xE9;plication de l&#x2019;ADN qui se produit dans les cellules. Le m&#xE9;lange r&#xE9;actionnel comprend une s&#xE9;quence d&#x2019;ADN matrice &#xE0; copier, une paire de courtes mol&#xE9;cules d&#x2019;ADN appel&#xE9;es amorces, des blocs de construction d&#x2019;ADN libres appel&#xE9;s des d&#xE9;soxynucl&#xE9;otides triphosphates (dNTP), et une enzyme sp&#xE9;cialis&#xE9;e d&#x2019;ADN polym&#xE9;rase. 
 
 La PCR implique une s&#xE9;rie d&#x2019;&#xE9;tapes &#xE0; des temp&#xE9;ratures &#xE9;lev&#xE9;es, n&#xE9;cessitant une enzyme d&#x2019;ADN polym&#xE9;rase qui est fonctionnelle &#xE0; de telles temp&#xE9;ratures. L&#x2019;ADN polym&#xE9;rase la plus couramment utilis&#xE9;e est la Taq polym&#xE9;rase, du nom de Thermus aquaticus, la bact&#xE9;rie &#xE0; partir de laquelle la polym&#xE9;rase a &#xE9;t&#xE9; isol&#xE9;e initialement. L&#x2019;ADN polym&#xE9;rase est incapable de synth&#xE9;tiser une mol&#xE9;cule d&#x2019;ADN &#xE0; partir de z&#xE9;ro, ou de novo. Au lieu de cela, l&#x2019;ADN polym&#xE9;rase ajoute aux courtes mol&#xE9;cules d&#x2019;ADN, appel&#xE9;es amorces, qui se lient &#xE0; la matrice d&#x2019;ADN par l&#x2019;appariement de bases compl&#xE9;mentaires. Les amorces fournissent un groupe hydroxyle libre en 3&#x2019; auquel l&#x2019;ADN polym&#xE9;rase peut attacher de nouveaux dNTP. Il existe quatre types de dNTP dans une PCR, un pour chaque nucl&#xE9;otide dans la mol&#xE9;cule d&#x2019;ADN : dATP, dCTP, dGTP et dTTP. 
 
 Chaque cycle PCR se compose de trois &#xE9;tapes : la d&#xE9;naturation, l&#x2019;hybridation et la synth&#xE8;se de l&#x2019;ADN. 
 
 <ol><li>D&#xE9;naturation Le cycle PCR est initi&#xE9; par le chauffage du m&#xE9;lange r&#xE9;actionnel &#xE0; une temp&#xE9;rature &#xE9;lev&#xE9;e, provoquant la s&#xE9;paration de l&#x2019;ADN double h&#xE9;lice en deux brins. Ce processus de &#x2018; fusion &#x201D; se produit habituellement &#xE0; une temp&#xE9;rature de 90 &#xB0;C &#xE0; 100 &#xB0;C.</li>
 <li>Hybridation Le m&#xE9;lange r&#xE9;actionnel est rapidement refroidi (habituellement &#xE0; 50 &#xB0;C&#x2013;65 &#xB0;C), permettant aux deux amorces de se lier &#xE0; leurs s&#xE9;quences compl&#xE9;mentaires sur les brins d&#x2019;ADN matrice.</li>
 <li>La synth&#xE8;se d&#x2019;ADN (l&#x2019;extension d&#x2019;amorce) Le m&#xE9;lange r&#xE9;actionnel est chauff&#xE9; &#xE0; nouveau, cette fois &#xE0; une temp&#xE9;rature (habituellement 60 &#xB0;C&#x2013;75 &#xB0;C) qui permet &#xE0; l&#x2019;ADN polym&#xE9;rase d&#x2019;&#xE9;tendre les amorces en ajoutant des dNTP qui s&#x2019;associent aux bases du brin matrice.</li>
 </ol>
 
 Une PCR typique implique 20 &#xE0; 40 cycles r&#xE9;p&#xE9;t&#xE9;s de ces trois &#xE9;tapes, se produisant dans le thermocycleur. Puisque le nombre de mol&#xE9;cules d&#x2019;ADN est doubl&#xE9; &#xE0; chaque cycle, l&#x2019;ADN est amplifi&#xE9; de fa&#xE7;on exponentielle.
 
 <h4>Les limites de la PCR</h4>
 
 Si le scientifique veut amplifier une portion sp&#xE9;cifique du g&#xE9;nome, il doit conna&#xEE;tre au moins une partie de la s&#xE9;quence d&#x2019;ADN cible pour concevoir des amorces appropri&#xE9;es. Un autre probl&#xE8;me potentiel est l&#x2019;hybridation non sp&#xE9;cifique des amorces &#xE0; des s&#xE9;quences d&#x2019;ADN partiellement similaires, conduisant &#xE0; l&#x2019;amplification d&#x2019;ADN non-cible. Ce probl&#xE8;me peut &#xEA;tre contr&#xF4;l&#xE9; en optimisant les conditions de r&#xE9;action. &#xC9;tant une m&#xE9;thode de d&#xE9;tection tr&#xE8;s sensible, la PCR est &#xE9;galement vuln&#xE9;rable &#xE0; la contamination, et m&#xEA;me des traces d&#x2019;ADN contaminant peuvent causer des r&#xE9;sultats trompeurs. Les polym&#xE9;rases d&#x2019;ADN utilis&#xE9;es dans la PCR peuvent &#xEA;tre sujettes &#xE0; des erreurs. Si une mutation se produit dans les premiers cycles, la plupart de l&#x2019;ADN amplifi&#xE9; portera la 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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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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