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

- [Instructor] El objetivo de la PCR, o

reacción en cadena de la polimerasa,

es amplificar una secuencia genética.

En este ejemplo, un gen de interés

será amplificado a
partir de ADN purificado.

Para realizar la amplificación,

se requiere una polimerasa
para sintetizar el nuevo ADN.

Un cebador, un trozo
corto de ADN monocatenario

que comparte homología
con el gen de interés,

también es necesario.

Finalmente, nucleótidos
individuales llamados

deoxinucleósidos 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 continuación, se enfría la reacción

y los cebadores se hibridarán
a su región homóloga.

Una vez unidos, la reacción se calienta

a una temperatura óptima
para la polimerasa.

La polimerasa reconoce entonces
el complejo de ADN cebador

y comienza la síntesis de la nueva cadena.

utilizando los dNTPs en la solución.

La reacción se calienta
y continúa como descrito

a través de ciclos adicionales.

Después del tercer ciclo,

hay ocho copias totales del gen.

Después del cuarto ciclo, 16 copias,

y en el quinto ciclo, 32 copias.

El número de ciclos continuará
creciendo exponencialmente.

Después de 30 ciclos, hay más

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>]

pcr - reacción en cadena de polimerasa

<h4>Descripción general</h4> La reacción en cadena de la polimerasa, o PCR, es una técnica ampliamente utilizada para copiar segmentos de ADN. Debido a la amplificación exponencial, la PCR puede producir millones o miles de millones de copias de ADN en tan sólo unas horas. En una reacción de PCR, una enzima ADN polimerasa resistente al calor amplifica el ADN original a través de una serie de cambios de temperatura dentro de una máquina automatizada llamada termociclador. <h4>La PCR es un método versátil que revolucionó la biología molecular</h4> Kary Mullis desarrolló la PCR en 1983, por la que recibió el Premio Nobel de Química en 1993. Al ser una forma relativamente rápida, económica y precisa de copiar una secuencia de ADN, la PCR se convirtió en una herramienta invaluable para numerosas aplicaciones, incluida la clonación molecular, la mutagénesis genética, la detección de patógenos, el análisis de la expresión genética, la cuantificación y secuenciación del ADN y el diagnóstico de enfermedades genéticas. <h4>El ciclo de reacción de la PCR</h4> La PCR imita el proceso natural de replicación del ADN que ocurre en las células. La mezcla de reacción incluye una secuencia de ADN modelo que se va a copiar, un par de moléculas de ADN cortas llamadas cebadores, componentes básicos de ADN libre llamados desoxinucleótidos 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 más utilizada es la polimerasa Taq, que lleva el nombre de Thermus aquaticus, la bacteria de la que se aisló inicialmente la polimerasa. La ADN polimerasa es incapaz de sintetizar una molécula de ADN desde cero o de novo. En cambio, la ADN polimerasa se suma a moléculas 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' hidroxilo libre al que la ADN polimerasa puede unir nuevos dNTP. Hay cuatro tipos de dNTP en una PCR, uno para cada nucleótido de la molécula de ADN: dATP, dCTP, dGTP y dTTP. Cada ciclo de PCR está formado por tres pasos: desnaturalización, hibridación y síntesis de ADN. <ol> <li>Desnaturalización. El ciclo de PCR se inicia calentando la mezcla de reacción a una temperatura alta, lo que provoca la separación de la doble hélice del ADN en dos hebras. Este proceso de "fusión" suele ocurrir a una temperatura de 90°C–100°C.</li> <li>Recocido. La mezcla de reacción se enfría rápidamente (normalmente entre 50 °C y 65 °C), lo que permite que los dos cebadores se unan a sus secuencias complementarias en las cadenas de ADN molde.</li> <li>Síntesis de ADN (extensión de cebadores). La mezcla de reacción se calienta nuevamente, esta vez a una temperatura (generalmente entre 60 °C–75°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 típica implica de 20 a 40 ciclos repetidos de estos tres pasos, que se realizan en el termociclador. Dado que el número de moléculas de ADN se duplica en cada ciclo, el ADN se amplifica exponencialmente. <h4>Limitaciones de la PCR</h4> Si el científico quiere amplificar un tramo específico del genoma, debe conocer al menos parte de la secuencia de ADN objetivo para diseñar cebadores apropiados. Otro problema potencial es la hibridación no específica de cebadores con secuencias de ADN parcialmente similares, lo que conduce a la amplificación de ADN no objetivo. Este problema se puede controlar optimizando las condiciones de reacción. Al ser un método de detección muy sensible, la PCR también es vulnerable a la contaminación, e incluso trazas de ADN contaminante pueden provocar resultados engañosos. Las ADN polimerasas utilizadas en la PCR pueden ser propensas a errores. Si ocurre una mutación dentro de los primeros ciclos, la mayor parte del ADN amplificado portará la mutación.

PCR - Reacción en Cadena de Polimerasa

Visión general

La reacción en cadena de la polimerasa, o PCR, es una técnica ampliamente utilizada para copiar segmentos de ADN. Debido a la amplificación exponencial, la PCR puede producir ...

Education

Educación

JoVE Core

Molecular Biology

Biología Molecular

Unit 2

Unidad 2

Studying DNA and RNA

Estudio del ADN y el ARN

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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

Recombinant DNA and Gene Cloning

Recombinant DNA and gene cloning are tools used to combine DNA from different sources. Scientists use them to isolate a gene, join it to a carrier DNA molecule, and make copies of that DNA. A plasmid is one common carrier used in this process. A plasmid is a small ring of DNA found in bacteria, and it can carry the inserted gene into a host cell. Once inside the cell, the recombinant DNA can be copied as the cell grows. This approach is important in biotechnology and genetic research. It...

dna isolation

Genomic DNA Extraction Methods

Genomic DNA extraction methods separate DNA from proteins, RNA, and other cell material. The process usually follows four main steps. First, the cell structure is disrupted with mechanical or chemical methods to make a cell lysate. Then the DNA is protected from damage during processing. After that, the soluble DNA is separated from cell debris. Finally, the purified DNA is eluted, or washed out, for later use. DNA isolation can follow two main approaches: solution-based methods or solid-phase...

dna agarose gel electrophoresis

Purifying DNA Bands from Agarose Gels

Agarose gel electrophoresis can be used to purify DNA bands after separation by size. In gel extraction, the DNA fragment of interest is cut from the gel and cleaned before later use. This is especially useful in cloning, where pure DNA is needed before ligation, the joining step that links DNA pieces together. In cloning experiments, both the insert and vector DNA fragments are first cut with restriction endonucleases. These fragments often contain enzymes, salts, and other contaminants. Such...

labeling dna probes

DNA Probe Labels for Detecting Targets

DNA probes are short DNA fragments with a reporter tag attached so they can be detected or purified. They bind to matching nucleic acid sequences by complementary base-pairing. That binding helps researchers recover or identify specific regions of DNA. Several reporter tags are commonly used to label DNA probes. These include radioisotopes, fluorophores, and small molecule binding partners such as biotin or digoxigenin. The label choice depends on the probe’s intended use and on the source of...

southern blot

Southern Blot for DNA Detection

Southern blotting is a DNA detection method used after agarose gel electrophoresis separates DNA fragments by size. A DNA ladder with fragments of known length is run beside the sample so the fragment size can be estimated. The blot adds another step when scientists need to confirm the sequence identity of the DNA. First, the target DNA is cut into smaller pieces with a restriction endonuclease and run on an agarose gel. The DNA is then denatured, which means the double strands are separated...

dna microarrays

Gene Chips for Comparing Cell Activity

DNA microarrays are gene chips used to compare gene activity in different conditions. They are high-throughput assays, which means they can analyze large amounts of data at once. They are also relatively inexpensive and can be automated, making them useful for genome-wide studies. These chips are built on glass or silica slides. Probe molecules are covalently attached to the surface through surface functionalization. In many cases, the slides are prepared by chemisorption of silanes to silica...

complementary dna

Making cDNA from RNA Templates

Complementary DNA, or cDNA, is made from an RNA template. It is a DNA copy of RNA, so it can help students study gene expression in a lab setting. cDNA is useful when scientists want to work with genes after they have been turned on in a cell. Because it is copied from RNA, it reflects which genes were active at the time the RNA was collected. This process links RNA and DNA in a simple way. It also gives students a clear view of how genetic information can be copied for analysis.

fish - fluorescent in-situ hybridization

FISH for Chromosome and Pathogen Detection

FISH is a DNA and RNA detection method used in cytogenetics and medical testing. FISH stands for fluorescence in situ hybridization. It uses labeled probes that bind to complementary DNA or RNA sequences on a chromosome or inside a cell. The method was developed in the early 1980s and became widely used because it can show specific genetic targets with high detail. In the past, probes were made by cloning or by reverse transcription of a DNA template. Today, probe oligonucleotides can also be...

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...

real time rt-pcr

Real-Time RT-PCR for Gene Expression Tracking

Real-time RT-PCR tracks gene expression by measuring how much target DNA is made during PCR. It starts with mRNA, which is converted into complementary DNA, or cDNA, by an enzyme called reverse transcriptase. The cDNA is then amplified by PCR so the target sequence can be measured. This method can be done in one tube as a single-step reaction or in two separate steps. During amplification, the amount of product is followed in real time with fluorescent dyes or sequence-specific probes linked...

race - rapid amplification of cdna ends

Finding cDNA Ends with RACE PCR

Rapid Amplification of cDNA Ends, or RACE, helps scientists capture the full-length cDNA from an mRNA sequence. It works by linking a known internal region to the unknown sequence at either the 5' end or the 3' end. The method is widely used when the missing end of a gene needs to be identified. RACE uses a gene-specific primer that binds to the known cDNA region. A hybrid primer then adds a set anchor sequence to the unknown end of the cDNA. After that, PCR amplifies the DNA segment between...

sanger sequencing

How Sanger Sequencing Reads DNA Fragments

Sanger sequencing reads DNA fragments by separating pieces of different lengths and decoding the order of bases. It is a key method in biology and is often used to study a cloned DNA fragment, a gene mutation, or even much larger genomes. Fredrick Sanger and his collaborators developed the chain-termination method in 1977. The method works by producing a mixture of DNA fragments with variable sizes. These fragments are then separated by capillary gel electrophoresis, which sorts them by length.

next-generation sequencing

DNA Sequencing Methods and Uses

DNA sequencing methods can read millions of DNA fragments at the same time. Next-generation sequencing made this process much faster and much less expensive than older methods. The first human genome project cost $2.7 billion and took 15 years. It was completed in 2003 after major international cooperation among research teams and funding agencies. To prepare DNA for next-generation sequencing, pure genomic DNA is first broken into smaller pieces. These pieces are used to build a sequencing...

rna-seq

RNA-Seq Workflow and Data Analysis

RNA sequencing, or RNA-Seq, is a high-throughput method for studying the transcriptome of a cell. The transcriptome is the full set of RNA molecules in a sample. Transcriptomics helps scientists understand how genomes work, how organisms develop, and how some diseases occur. Before RNA-Seq, researchers often used microarray methods and Sanger sequencing to study RNA. Microarrays had limited coverage and depended on already knowing the genome sequence. Sanger sequencing was low-throughput,...

genome annotation and assembly

How Genome Assemblies Are Updated

Genome assembly and genome annotation are key steps in genome analysis. Genome assembly puts DNA sequencing data back in the correct order to make a close copy of the original genome. Genome annotation then identifies functional parts on that assembled genome. Assembling a genome is not simple. In human populations, gene copy number can vary, and repeated DNA sequences can make the process harder. Even so, the physical location of human genes stays constant. Bacterial genomes can be more...

restriction enzymes

DNA Cutting Sites and Bacterial Defense

Restriction enzymes are bacterial proteins that cut DNA at specific sequence sites. They recognize palindromic DNA sequences, also called inverted repeats, and cut the DNA in a sequence-specific way. These sites are often found in regions with important functions, such as the origin of replication, gene operator sites, and transcription termination signals. Bacteria protect their own DNA by methylating these same sites. Methylation means adding chemical marks that keep the restriction enzyme...

maxam-gilbert sequencing

Chemical Cleavage DNA Sequencing

Chemical cleavage DNA sequencing uses specific chemicals to cut DNA at known sites. Allan Maxam and Walter Gilbert described this method in the same year that the Sanger sequencing method was discovered. It reads the DNA sequence by breaking purified DNA into fragments and then studying the fragment pattern. The method depends on chemicals that cleave DNA at specific bases. The resulting fragments are separated by electrophoresis, which is a way to sort DNA pieces by size using an electric...

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 Biología Molecular presenta los procesos moleculares de la célula a través de lecciones claras y concisas de animaciones audiovisuales. Además, en los vídeos de científicos en acción, se muestran aplicaciones de estos conceptos mediante experimentos reales en laboratorios especializados.

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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Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
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BIO1.ETS2.3

PCR: DNA Amplification for Genetics Labs

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