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

- [Istruttore] L&#x27;obiettivo della RCP,

reazione a catena della polimerasi

&#xE8; di amplificare una sequenza genetica.

In questo esempio,

un gene di interesse sar&#xE0; amplificato

dal DNA purificato.

Per eseguire l&#x27;amplificazione,

&#xE8; necessaria una polimerasi
per sintetizzare il nuovo DNA.

Anche un primer, breve pezzo
di DNA a filamento singolo

che condivide l&#x27;omologia
con il gene di interesse,

&#xE8; necessario.

Infine, singoli nucleotidi,

chiamati disossinucleotidi
trifosfati o dNTP,

sono usati per creare il nuovo filone.

Il primo passo nella PCR

&#xE8; riscaldare la miscela,

che denatura il DNA.

Successivamente la
reazione viene raffreddata,

e i primer si ricopriranno
nella loro regione omologa.

Una volta legati, la
reazione viene riscaldata

a una temperatura ottimale
per la polimerasi.

La polimerasi quindi riconosce

il complesso del DNA dell&#x27;innesco,

e inizia la sintesi del nuovo filone,

utilizzando i dNTP nella soluzione.

La reazione viene quindi riscaldata

e procede come descritto,

attraverso ulteriori cicli.

Dopo il terzo ciclo,

ci sono otto copie totali del gene.

Dopo il quarto ciclo, 16 copie.

E il quinto ciclo, 32 copie.

Il numero di copie continua a
crescere in modo esponenziale.

Dopo 30 cicli, ci sono poco
pi&#xF9; di 1 miliardo di copie.

Garibyan, Lilit e Nidhi Avashia. "Tecniche di ricerca rese semplici: reazione a catena della polimerasi (PCR)." The Journal of Investigative Dermatology 133, n. 3 (marzo 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Sorgente</a>]

Pray, Leslie A. "La rivoluzione biotecnologica: PCR e l'uso della trascrittasi inversa per clonare geni espressi". Educazione alla natura 1, n. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Sorgente</a>]

<h4>Panoramica</h4>

La reazione a catena della polimerasi, o PCR, &#xE8; una tecnica ampiamente utilizzata per copiare segmenti di DNA. Grazie all&#x27;amplificazione esponenziale, la PCR pu&#xF2; produrre milioni o miliardi di copie di DNA in poche ore. In una reazione PCR, un enzima di polimerasi del DNA resistente al calore amplifica il DNA originale attraverso una serie di variazioni di temperatura all&#x27;interno di una macchina automatizzata chiamata termociclo.

<h4>PCR &#xE8; un metodo versatile che ha rivoluzionato la biologia molecolare</h4>

Kary Mullis ha sviluppato la PCR nel 1983, per la quale &#xE8; stato insignito del Premio Nobel per la chimica nel 1993. Essendo un modo relativamente veloce, economico e preciso di copiare una sequenza di DNA, la PCR &#xE8; diventata uno strumento inestimabile per numerose applicazioni, tra cui la clonazione molecolare, la mutagenesi genica, il rilevamento di patogeni, l&#x27;analisi dell&#x27;espressione genica, la quantificazione del DNA e il sequenziamento e la diagnosi delle malattie genetiche.

<h4>Il ciclo di reazione PCR</h4>

La PCR imita il processo di replicazione del DNA naturale che si verifica nelle cellule. La miscela di reazione include una sequenza di DNA modello da copiare, una coppia di molecole di DNA corto chiamate primer, blocchi di costruzione di DNA libero chiamati triplofati deossinucleotidi (dNTP) e un enzima specializzato di polimerasi del DNA.

La PCR comporta una serie di passaggi ad alte temperature, che richiedono un enzima di polimerasi del DNA che &#xE8; funzionale a tali temperature. La polimerasi del DNA pi&#xF9; comunemente utilizzata &#xE8; la polimerasi Taq, dal nome della Thermus aquaticus, il batterio da cui la polimerasi &#xE8; stata inizialmente isolata. La polimerasi del DNA non &#xE8; in grado di sintetizzare una molecola di DNA da zero, o de novo. Invece, la polimerasi del DNA si aggiunge alle molecole di DNA corto, chiamate primer, che si legano al modello di DNA attraverso l&#x27;accoppiamento di base complementare. I primer forniscono un gruppo di idrossile gratuito da 3&#x27; a cui la polimerasi del DNA pu&#xF2; collegare nuovi dNTP. Ci sono quattro tipi di dNTP in una PCR, uno per ogni nucleotide nella molecola di DNA: dATP, dCTP, dGTP e dTTP.

Ogni ciclo di PCR &#xE8; costituito da tre fasi: Denaturazione, Annealing e Sintesi del DNA.

<ol><li>Denaturazione. Il ciclo PCR viene avviato riscaldando la miscela di reazione ad una temperatura elevata, causando la separazione del DNA doppia elica in due fili. Questo processo di &#x22;fusione&#x22; di solito si verifica ad una temperatura di 90-100 gradi centigradi.</li>
	<li>Ricottura. La miscela di reazione viene raffreddata rapidamente (di solito a 50oC&#x2013;65oC), permettendo ai due primer di legarsi alle loro sequenze complementari sui filamenti di DNA del modello.</li>
	<li>Sintesi del DNA (Estensione Primer). La miscela di reazione viene riscaldata di nuovo, questa volta ad una temperatura (di solito 60-75gradi) che consente alla polimerasi del DNA di estendere le primer aggiungendo dNTP che si accoppiano con le basi nel filo modello.</li>
</ol>Una PCR tipica coinvolge 20-40 cicli ripetuti di questi tre passaggi, che si verificano nel termociclore. Poich&#xE9; il numero di molecole di DNA &#xE8; raddoppiato in ogni ciclo, il DNA viene amplificato in modo esponenziale.

<h4>Limitazioni della PCR</h4>

Se lo scienziato vuole amplificare un tratto specifico del genoma, lo scienziato deve conoscere almeno una parte della sequenza del DNA bersaglio per progettare i primer appropriati. Un altro potenziale problema &#xE8; l&#x27;annessione non specifica dei primer a sequenze di DNA parzialmente simili, portando all&#x27;amplificazione del DNA non bersaglio. Questo problema pu&#xF2; essere controllato ottimizzando le condizioni di reazione. Essendo un metodo di rilevamento altamente sensibile, la PCR &#xE8; anche vulnerabile alla contaminazione e anche tracce di DNA contaminato possono causare risultati fuorvianti. Le polimerasi del DNA utilizzate nella PCR possono essere soggette a errori. Se una mutazione avviene entro i primi cicli, la maggior parte del DNA amplificato porter&#xE0; la mutazione.

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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