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

- [Instructor] El objetivo de la PCR, o

reacci&#xF3;n en cadena de la polimerasa,

es amplificar una secuencia gen&#xE9;tica.

En este ejemplo, un gen de inter&#xE9;s

ser&#xE1; amplificado a
partir de ADN purificado.

Para realizar la amplificaci&#xF3;n,

se requiere una polimerasa
para sintetizar el nuevo ADN.

Un cebador, un trozo
corto de ADN monocatenario

que comparte homolog&#xED;a
con el gen de inter&#xE9;s,

tambi&#xE9;n es necesario.

Finalmente, nucle&#xF3;tidos
individuales llamados

deoxinucle&#xF3;sidos trifosfatos, o dNTPs,

se utilizan para hacer la nueva hebra.

El primer paso en la PCR
es calentar la mezcla,

que desnaturaliza el ADN.

A continuaci&#xF3;n, se enfr&#xED;a la reacci&#xF3;n

y los cebadores se hibridar&#xE1;n
a su regi&#xF3;n hom&#xF3;loga.

Una vez unidos, la reacci&#xF3;n se calienta

a una temperatura &#xF3;ptima
para la polimerasa.

La polimerasa reconoce entonces
el complejo de ADN cebador

y comienza la s&#xED;ntesis de la nueva cadena.

utilizando los dNTPs en la soluci&#xF3;n.

La reacci&#xF3;n se calienta
y contin&#xFA;a como descrito

a trav&#xE9;s de ciclos adicionales.

Despu&#xE9;s del tercer ciclo,

hay ocho copias totales del gen.

Despu&#xE9;s del cuarto ciclo, 16 copias,

y en el quinto ciclo, 32 copias.

El n&#xFA;mero de ciclos continuar&#xE1;
creciendo exponencialmente.

Despu&#xE9;s de 30 ciclos, hay m&#xE1;s

de mil millones de copias.

Garibyan, Lilit y Nidhi Avashia. "Técnicas de investigación simplificadas: reacción en cadena de la polimerasa (PCR)". The Journal of Investigative Dermatology 133, no. 3 (marzo de 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Fuente</a>]

Pray, Leslie A. "La revolución de la biotecnología: PCR y el uso de la transcriptasa inversa para clonar genes expresados". Nature Education 1, no. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Fuente</a>]

<h4>Descripci&#xF3;n general</h4> La reacci&#xF3;n en cadena de la polimerasa, o PCR, es una t&#xE9;cnica ampliamente utilizada para copiar segmentos de ADN. Debido a la amplificaci&#xF3;n exponencial, la PCR puede producir millones o miles de millones de copias de ADN en tan s&#xF3;lo unas horas. En una reacci&#xF3;n de PCR, una enzima ADN polimerasa resistente al calor amplifica el ADN original a trav&#xE9;s de una serie de cambios de temperatura dentro de una m&#xE1;quina automatizada llamada termociclador. <h4>La PCR es un m&#xE9;todo vers&#xE1;til que revolucion&#xF3; la biolog&#xED;a molecular</h4> Kary Mullis desarroll&#xF3; la PCR en 1983, por la que recibi&#xF3; el Premio Nobel de Qu&#xED;mica en 1993. Al ser una forma relativamente r&#xE1;pida, econ&#xF3;mica y precisa de copiar una secuencia de ADN, la PCR se convirti&#xF3; en una herramienta invaluable para numerosas aplicaciones, incluida la clonaci&#xF3;n molecular, la mutag&#xE9;nesis gen&#xE9;tica, la detecci&#xF3;n de pat&#xF3;genos, el an&#xE1;lisis de la expresi&#xF3;n gen&#xE9;tica, la cuantificaci&#xF3;n y secuenciaci&#xF3;n del ADN y el diagn&#xF3;stico de enfermedades gen&#xE9;ticas. <h4>El ciclo de reacci&#xF3;n de la PCR</h4> La PCR imita el proceso natural de replicaci&#xF3;n del ADN que ocurre en las c&#xE9;lulas. La mezcla de reacci&#xF3;n incluye una secuencia de ADN modelo que se va a copiar, un par de mol&#xE9;culas de ADN cortas llamadas cebadores, componentes b&#xE1;sicos de ADN libre llamados desoxinucle&#xF3;tidos trifosfatos (dNTP) y una enzima ADN polimerasa especializada. La PCR implica una serie de pasos a altas temperaturas, que requieren una enzima ADN polimerasa que sea funcional a dichas temperaturas. La ADN polimerasa m&#xE1;s utilizada es la polimerasa Taq, que lleva el nombre de Thermus aquaticus, la bacteria de la que se aisl&#xF3; inicialmente la polimerasa. La ADN polimerasa es incapaz de sintetizar una mol&#xE9;cula de ADN desde cero o de novo. En cambio, la ADN polimerasa se suma a mol&#xE9;culas cortas de ADN, llamadas cebadores, que se unen a la plantilla de ADN mediante un emparejamiento de bases complementarias. Los cebadores proporcionan un grupo 3&#x27; hidroxilo libre al que la ADN polimerasa puede unir nuevos dNTP. Hay cuatro tipos de dNTP en una PCR, uno para cada nucle&#xF3;tido de la mol&#xE9;cula de ADN: dATP, dCTP, dGTP y dTTP. Cada ciclo de PCR est&#xE1; formado por tres pasos: desnaturalizaci&#xF3;n, hibridaci&#xF3;n y s&#xED;ntesis de ADN. <ol> <li>Desnaturalizaci&#xF3;n. El ciclo de PCR se inicia calentando la mezcla de reacci&#xF3;n a una temperatura alta, lo que provoca la separaci&#xF3;n de la doble h&#xE9;lice del ADN en dos hebras. Este proceso de &#x22;fusi&#xF3;n&#x22; suele ocurrir a una temperatura de 90&#xB0;C&#x2013;100&#xB0;C.</li> <li>Recocido. La mezcla de reacci&#xF3;n se enfr&#xED;a r&#xE1;pidamente (normalmente entre 50 &#xB0;C y 65 &#xB0;C), lo que permite que los dos cebadores se unan a sus secuencias complementarias en las cadenas de ADN molde.</li> <li>S&#xED;ntesis de ADN (extensi&#xF3;n de cebadores). La mezcla de reacci&#xF3;n se calienta nuevamente, esta vez a una temperatura (generalmente entre 60 &#xB0;C&#x2013;75&#xB0;C) que permite que la ADN polimerasa extienda los cebadores agregando dNTP que se emparejan con las bases en la cadena plantilla.</li> </ol> Una PCR t&#xED;pica implica de 20 a 40 ciclos repetidos de estos tres pasos, que se realizan en el termociclador. Dado que el n&#xFA;mero de mol&#xE9;culas de ADN se duplica en cada ciclo, el ADN se amplifica exponencialmente. <h4>Limitaciones de la PCR</h4> Si el cient&#xED;fico quiere amplificar un tramo espec&#xED;fico del genoma, debe conocer al menos parte de la secuencia de ADN objetivo para dise&#xF1;ar cebadores apropiados. Otro problema potencial es la hibridaci&#xF3;n no espec&#xED;fica de cebadores con secuencias de ADN parcialmente similares, lo que conduce a la amplificaci&#xF3;n de ADN no objetivo. Este problema se puede controlar optimizando las condiciones de reacci&#xF3;n. Al ser un m&#xE9;todo de detecci&#xF3;n muy sensible, la PCR tambi&#xE9;n es vulnerable a la contaminaci&#xF3;n, e incluso trazas de ADN contaminante pueden provocar resultados enga&#xF1;osos. Las ADN polimerasas utilizadas en la PCR pueden ser propensas a errores. Si ocurre una mutaci&#xF3;n dentro de los primeros ciclos, la mayor parte del ADN amplificado portar&#xE1; la mutaci&#xF3;n.

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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