Name: pcr: dna amplification for genetics labs
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 1 min 32 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

PCR - Polymerase Chain Reaction

Le but de la PCR,Réaction en chaîne de la polyméraseest d'amplifier une séquence génétique. 
Dans cet exemple,un gène d'intérêt sera amplifiéà partir d'ADN purifié. 
Pour effectuer l'amplification,une polymérase est nécessairepour synthétiser le nouvel ADN. 
Une amorce, un petit morceau d'ADN monocaténairequi partage une homologie avec le gène d'intérêt,est également nécessaire. 
Enfin, les nucléotides simples,appelés triphosphates de désoxynucléotides ou dNTP,sont utilisés pour fabriquer le nouveau brin. 
La première étape de la PCRest de chauffer le mélange,qui dénature l'ADN. 
Ensuite, la réaction est refroidie,et les amorces seront appareillées à leur région homologue. 
Une fois liée, la réaction est chaufféeà une température optimale pour la polymérase. 
La polymérase reconnaît alorsle complexe de l'amorce d'ADN,et commence la synthèse du nouveau brin,en utilisant les dNTP de la solution. 
La réaction est ensuite chaufféeet procède tel que c'est décrit,d'un cycle à l'autre. 
Après le troisième cycle,il y a 8 copies du gène au total. 
Après le quatrième cycle, 16 copies. 
Et cinquième cycle, 32 copies. 
Le nombre d'exemplaires continuera de croîtrede façon exponentielle. 
Après 30 cycles,il y a un peu plus d'un milliard d'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>]

pcr - réaction en chaîne par polymérase

<h4>Aperçu</h4>
 
 La réaction en chaîne par polymérase, ou PCR, est une technique largement utilisée pour copier des segments d’ADN. En raison de l’amplification exponentielle, la PCR peut produire des millions ou des milliards de copies d’ADN en quelques heures. Dans une réaction PCR, une enzyme d’ADN polymérase résistante à la chaleur amplifie l’ADN d’origine par le biais d’une série de changements de température à l’intérieur d’une machine automatisée appelée thermocycleur. 
 
 <h4>La PCR est une méthode polyvalente qui a révolutionné la biologie moléculaire</h4>
 
 Kary Mullis a mis au point la PCR en 1983, pour laquelle il a reçu le prix Nobel de chimie en 1993. Étant un moyen relativement rapide, peu coûteux et précis de copier une séquence d’ADN, la PCR est devenu un outil précieux pour de nombreuses applications, y compris le clonage moléculaire, la mutagenèse des gènes, la détection des agents pathogènes, l’analyse de l’expression génique, la mesure de la quantité d’ADN et le séquençage ainsi que le diagnostic des maladies génétiques.
 
 <h4>Le cycle des réactions PCR</h4>
 
 La PCR imite le processus naturel de réplication de l’ADN qui se produit dans les cellules. Le mélange réactionnel comprend une séquence d’ADN matrice à copier, une paire de courtes molécules d’ADN appelées amorces, des blocs de construction d’ADN libres appelés des désoxynucléotides triphosphates (dNTP), et une enzyme spécialisée d’ADN polymérase. 
 
 La PCR implique une série d’étapes à des températures élevées, nécessitant une enzyme d’ADN polymérase qui est fonctionnelle à de telles températures. L’ADN polymérase la plus couramment utilisée est la Taq polymérase, du nom de Thermus aquaticus, la bactérie à partir de laquelle la polymérase a été isolée initialement. L’ADN polymérase est incapable de synthétiser une molécule d’ADN à partir de zéro, ou de novo. Au lieu de cela, l’ADN polymérase ajoute aux courtes molécules d’ADN, appelées amorces, qui se lient à la matrice d’ADN par l’appariement de bases complémentaires. Les amorces fournissent un groupe hydroxyle libre en 3’ auquel l’ADN polymérase peut attacher de nouveaux dNTP. Il existe quatre types de dNTP dans une PCR, un pour chaque nucléotide dans la molécule d’ADN : dATP, dCTP, dGTP et dTTP. 
 
 Chaque cycle PCR se compose de trois étapes : la dénaturation, l’hybridation et la synthèse de l’ADN. 
 
 <ol><li>Dénaturation Le cycle PCR est initié par le chauffage du mélange réactionnel à une température élevée, provoquant la séparation de l’ADN double hélice en deux brins. Ce processus de ‘ fusion ” se produit habituellement à une température de 90 °C à 100 °C.</li>
 <li>Hybridation Le mélange réactionnel est rapidement refroidi (habituellement à 50 °C–65 °C), permettant aux deux amorces de se lier à leurs séquences complémentaires sur les brins d’ADN matrice.</li>
 <li>La synthèse d’ADN (l’extension d’amorce) Le mélange réactionnel est chauffé à nouveau, cette fois à une température (habituellement 60 °C–75 °C) qui permet à l’ADN polymérase d’étendre les amorces en ajoutant des dNTP qui s’associent aux bases du brin matrice.</li>
 </ol>
 
 Une PCR typique implique 20 à 40 cycles répétés de ces trois étapes, se produisant dans le thermocycleur. Puisque le nombre de molécules d’ADN est doublé à chaque cycle, l’ADN est amplifié de façon exponentielle.
 
 <h4>Les limites de la PCR</h4>
 
 Si le scientifique veut amplifier une portion spécifique du génome, il doit connaître au moins une partie de la séquence d’ADN cible pour concevoir des amorces appropriées. Un autre problème potentiel est l’hybridation non spécifique des amorces à des séquences d’ADN partiellement similaires, conduisant à l’amplification d’ADN non-cible. Ce problème peut être contrôlé en optimisant les conditions de réaction. Étant une méthode de détection très sensible, la PCR est également vulnérable à la contamination, et même des traces d’ADN contaminant peuvent causer des résultats trompeurs. Les polymérases d’ADN utilisées dans la PCR peuvent être sujettes à des erreurs. Si une mutation se produit dans les premiers cycles, la plupart de l’ADN amplifié portera la mutation.

PCR - Réaction en chaîne par polymérase

Aperçu

La réaction en chaîne par polymérase, ou PCR, est une technique largement utilisée pour copier des segments d’ADN. En raison de l’amplification exponentielle, la PCR peut produire ...

Education

Enseignement

JoVE Core

Molecular Biology

Biologie moléculaire

Unit 2

Unité 2

Studying DNA and RNA

L'étude de l'ADN et de l'ARN

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

pcr: dna amplification for genetics labs

PCR is a DNA amplification method used in genetics labs. PCR stands for polymerase chain reaction. It copies a specific DNA segment so scientists can study small amounts of genetic material.

The process uses repeated heating and cooling steps. These steps let the DNA strands separate, primers bind to the target region, and a DNA polymerase build new strands. With each cycle, the target DNA can increase in amount.

PCR is a core technique in molecular biology and many school-level biology topics. It helps students connect DNA structure with laboratory methods used to analyze genetic information.

PCR: DNA Amplification for Genetics Labs

recombinant dna

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southern blot

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pcr - polymerase chain reaction

PCR is a DNA amplification method used in genetics labs. PCR stands for polymerase chain reaction. It copies a specific DNA segment so scientists can study small amounts of genetic material. The process uses repeated heating and cooling steps. These steps let the DNA strands separate, primers bind to the target region, and a DNA polymerase build new strands. With each cycle, the target DNA can increase in amount. PCR is a core technique in molecular biology and many school-level biology...

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key terms and concepts

Learn how PCR amplifies a specific DNA segment through heating, cooling, primers, and DNA polymerase in genetics and molecular biology labs.

PCR: DNA Amplification for Genetics Labs | JoVE K12

molecular biology

JoVE Core Biologie Moléculaire illustre les processus moléculaires au sein de la cellule grâce à des leçons vidéos claires et concises. Les applications de ces concepts sont ensuite démontrées grâce à des expériences concrètes en laboratoire.

Florida Department of Education

Life Science

SC.912.L.16

SC.912.L.16.12

Texas Essential Knowledge and Skills

BIO.7

BIO.7.D

myJoVE Corporation

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BIO1.ETS2.3

PCR: DNA Amplification for Genetics Labs

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