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

O objetivo da PCR, rea&#xE7;&#xE3;o em cadeia da polimerase, &#xE9; amplificar uma sequ&#xEA;ncia gen&#xE9;tica. Neste exemplo, um gene de interesse ser&#xE1; ampliado a partir de DNA purificado. Para realizar a amplifica&#xE7;&#xE3;o, uma polimerase &#xE9; necess&#xE1;ria para sintetizar o novo DNA.Um primer, uma pequena pe&#xE7;a de DNA de fita simples que compartilha homologia com o gene de interesse, tamb&#xE9;m &#xE9; necess&#xE1;rio. Finalmente, nucleot&#xED;deos &#xFA;nicos chamados trifosfatos de desoxinucleotide, ou dNTPs, s&#xE3;o usados para fazer a nova vertente. A primeira etapa da PCR &#xE9; aquecer a mistura, que desnatura o DNA.Em seguida, a rea&#xE7;&#xE3;o &#xE9; resfriada e os primers ir&#xE3;o se alinhar para sua regi&#xE3;o hom&#xF3;loga. Uma vez ligada, a rea&#xE7;&#xE3;o &#xE9; aquecida a uma temperatura &#xF3;tima para a polimerase. A polimerase ent&#xE3;o reconhece o complexo primer de DNA e come&#xE7;a a s&#xED;ntese da nova vertente, usando os dNTPs na solu&#xE7;&#xE3;o.A rea&#xE7;&#xE3;o &#xE9; ent&#xE3;o aquecida e prossegue como descrito em em ciclos adicionais. Ap&#xF3;s o terceiro ciclo, existem, no total, oito c&#xF3;pias do gene. Ap&#xF3;s o quarto ciclo, dezasseis exemplares.E no quinto ciclo, trinta e duas c&#xF3;pias. O n&#xFA;mero de c&#xF3;pias ir&#xE1; continuar a crescer exponencialmente. Depois de trinta ciclos, existem pouco mais de um bilh&#xE3;o de c&#xF3;pias.

Garibyan, Lilit e Nidhi Avashia. "Técnicas de pesquisa simplificadas: reação em cadeia da polimerase (PCR)." The Journal of Investigative Dermatology 133, nº 3 (março de 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Fonte</a>]

Pray, Leslie A. "A Revolução da Biotecnologia: PCR e o Uso da Transcriptase Reversa para Clonar Genes Expressos." 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">Fonte</a>]

<h4>Vis&#xE3;o Geral</h4>
 
 A rea&#xE7;&#xE3;o em cadeia da polimerase, ou PCR, &#xE9; uma t&#xE9;cnica amplamente utilizada para copiar segmentos de DNA. Devido a amplifica&#xE7;&#xE3;o exponencial, o PCR pode produzir milh&#xF5;es ou bili&#xF5;es de c&#xF3;pias de DNA em apenas algumas horas. Em uma rea&#xE7;&#xE3;o de PCR, uma enzima DNA polimerase resistente ao calor amplifica o DNA original atrav&#xE9;s de uma s&#xE9;rie de altera&#xE7;&#xF5;es de temperatura dentro de uma m&#xE1;quina automatizada chamada termociclador.
 
 <h4>O PCR &#xE9; um M&#xE9;todo Vers&#xE1;til que Revolucionou a Biologia Molecular</h4>
 
 Kary Mullis desenvolveu o PCR em 1983, pelo qual recebeu o Pr&#xE9;mio Nobel da Qu&#xED;mica em 1993. Sendo uma maneira relativamente r&#xE1;pida, barata e precisa de copiar uma sequ&#xEA;ncia de DNA, o PCR tornou-se uma ferramenta inestim&#xE1;vel para in&#xFA;meras aplica&#xE7;&#xF5;es, incluindo clonagem molecular, mutag&#xE9;nese gen&#xE9;tica, detec&#xE7;&#xE3;o de angentes patog&#xE9;nicos, an&#xE1;lise de express&#xE3;o gen&#xE9;tica, quantifica&#xE7;&#xE3;o e sequenciamento de DNA e diagn&#xF3;stico de doen&#xE7;as gen&#xE9;ticas.
 
 <h4>O Ciclo de Rea&#xE7;&#xE3;o do PCR</h4>
 
 O PCR imita o processo natural de replica&#xE7;&#xE3;o do DNA que ocorre nas c&#xE9;lulas. A mistura da rea&#xE7;&#xE3;o inclui uma sequ&#xEA;ncia de DNA molde a ser copiada, um par de mol&#xE9;culas de DNA curtas chamadas primers, blocos de constru&#xE7;&#xE3;o de DNA livres chamados deoxinucle&#xF3;tidos trifosfato (dNTPs), e uma enzima DNA polimerase especializada.
 
 O PCR envolve uma s&#xE9;rie de passos a altas temperaturas, exigindo uma enzima DNA polimerase que seja funcional a tais temperaturas. A DNA polimerase mais usada &#xE9; a Taq polimerase, nomeada em homenagem a Thermus aquaticus, a bact&#xE9;ria da qual a polimerase foi inicialmente isolada. A DNA polimerase &#xE9; incapaz de sintetizar uma mol&#xE9;cula de DNA do zero, ou de novo. Em vez disso, a DNA polimerase complementa mol&#xE9;culas de DNA curtas, chamadas primers, que se ligam ao molde de DNA atrav&#xE9;s do emparelhamento complementar de bases. Os primers fornecem um grupo hidroxilo 3&#x2019; livre ao qual a DNA polimerase pode anexar novos dNTPs. Existem quatro tipos de dNTPs em um PCR, um para cada nucle&#xF3;tido na mol&#xE9;cula de DNA: dATP, dCTP, dGTP e dTTP.
 
 Cada ciclo de PCR consiste em tr&#xEA;s passos: Desnatura&#xE7;&#xE3;o, Anelamento e S&#xED;ntese de DNA.
 
 <ol>
 <li>Desnatura&#xE7;&#xE3;o. O ciclo de PCR &#xE9; iniciado aquecendo a mistura de rea&#xE7;&#xE3;o a uma alta temperatura, causando a separa&#xE7;&#xE3;o da dupla h&#xE9;lice do DNA em duas cadeias. Este processo de &#x201C;fus&#xE3;o&#x201D; geralmente ocorre a uma temperatura de 90&#xB0;C&#xAD;&#xAD;&#x2013;100&#xB0;C.</li>
 <li>Anelamento. A mistura de rea&#xE7;&#xE3;o &#xE9; rapidamente arrefecida (geralmente para 50&#xB0;C&#x2013;65&#xB0;C), permitindo que os dois primers se liguem &#xE0;s suas sequ&#xEA;ncias complementares nas cadeias de DNA do molde.</li>
 <li>S&#xED;ntese de DNA (Extens&#xE3;o de Primers). A mistura de rea&#xE7;&#xE3;o &#xE9; aquecida novamente, desta vez a uma temperatura (geralmente 60&#xB0;C&#x2013;75&#xB0;C) que permite que a DNA polimerase estenda os primers adicionando dNTPs que emparelham com as bases da cadeia molde.</li>
 </ol>
 
 Um PCR t&#xED;pico envolve 20-40 ciclos repetidos desses tr&#xEA;s passos, que ocorrem no termociclador. Como o n&#xFA;mero de mol&#xE9;culas de DNA &#xE9; duplicado em cada ciclo, o DNA &#xE9; amplificado exponencialmente.
 
 <h4>Limita&#xE7;&#xF5;es do PCR</h4>
 
 Se o cientista quiser amplificar uma por&#xE7;&#xE3;o espec&#xED;fica do genoma, o cientista deve saber pelo menos parte da sequ&#xEA;ncia de DNA alvo para criar primers apropriados. Outro problema potencial &#xE9; o anelamento n&#xE3;o espec&#xED;fico de primers a sequ&#xEA;ncias de DNA parcialmente semelhantes, levando &#xE0; amplifica&#xE7;&#xE3;o de DNA n&#xE3;o alvo. Este problema pode ser controlado optimizando as condi&#xE7;&#xF5;es da rea&#xE7;&#xE3;o. Sendo um m&#xE9;todo de detec&#xE7;&#xE3;o altamente sens&#xED;vel, o PCR tamb&#xE9;m &#xE9; vulner&#xE1;vel a contamina&#xE7;&#xE3;o, e at&#xE9; mesmo vest&#xED;gios de DNA contaminante podem originar resultados enganadores. As DNA polimerases usados no PCR podem ser propensas a erros. Se uma muta&#xE7;&#xE3;o acontecer nos primeiros ciclos, a maior parte do DNA amplificado carregar&#xE1; a muta&#xE7;&#xE3;o.

Overview

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce ...

https://cloudfront.jove.com/files/ftp_upload/10819/10819_article_thumb_10819.jpg

Education

JoVE Core

Biology

Biologia

Genetics

Genética

Biotechnology

Biotecnologia

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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

what is genetic engineering?

How Scientists Edit DNA for New Traits

Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

dna isolation

Steps in DNA Extraction

DNA extraction is the process of removing and purifying DNA from cells. It is used in many biotechnology and research applications, including molecular cloning. The main goal is to free DNA from the cell and prepare it for later study or use. The first step is cell lysis, which means breaking cells open. Researchers may use a homogenizer, sonicator, or bead beater to physically lyse the cells. They also often add detergent to chemically disrupt the lipid-based cell membranes. This helps...

recombinant dna technology

Recombinant DNA in Cloning and GMOs

Recombinant DNA is made by joining DNA from different sources in the lab. Scientists often use it to clone genes and study them in cells that are easy to handle, such as bacteria. Organisms that carry recombinant DNA are called genetically modified organisms, or GMOs. This technology gives scientists new tools for research, medicine, and agriculture. To build recombinant DNA, researchers place a gene of interest into a vector, which is a DNA carrier that moves foreign DNA into a host cell. The...

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...

complementary dna

From mRNA to cDNA for Gene Studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

antibiotic selection

Antibiotic Resistance in Cloning

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world. Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them.

transgenic organisms

Creating Transgenic Plants and Animals

Transgenic organisms are genetically engineered to carry a transgene, which is a gene from a different species, in their genome. The inserted gene may be a new gene for that organism or a different version of one it already has. Scientists usually make transgenes with recombinant DNA and DNA cloning methods. Researchers use transgenic bacteria, plants, and animals to answer biological questions and to build useful tools. To make a transgenic organism, they first choose a transgenesis method,...

in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes. A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through...

crispr

CRISPR-Cas9 Gene Editing in Action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

gene therapy

Gene Delivery for Serious Diseases

Gene therapy is a technique used to treat serious disease by adding a gene to a person’s cells. The added gene may be a healthy copy of a mutated gene. It may also be a different gene that inactivates or compensates for the disease-causing gene. One example is severe combined immunodeficiency, or SCID, caused by a mutation in the gene for adenosine deaminase. In that case, a working copy of the gene can be inserted so the patient’s cells make the enzyme again. This can cure the disease in some...

embryonic stem cells

How Embryonic Stem Cells Become Specialized

Embryonic stem cells are pluripotent cells that have not yet specialized. Pluripotent means they can give rise to many different cell types in the body. Because of this, they are useful in science and medicine for making cells needed in research or for replacing cells lost to damage or disease. These cells come from the inner cell mass of a blastocyst, which is an early embryo about 3 to 5 days after fertilization in humans. At this stage, the embryo has not yet implanted in the uterus. Human...

adult stem cells

Adult Stem Cells in the Small Intestine

Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

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

JoVE Core series brings biology to life through over 300 concise and easy-to-understand animated video lessons that explain key concepts in biology, plus more than 150 scientist-in-action videos that show actual research experiments conducted in today’s laboratories.

biology

A coleção JoVE Core dá vida à biologia através de mais de 300 vídeos de animação concisos e fáceis de entender que explicam conceitos-chave de biologia, além de mais de 150 vídeos de Cientistas em ação que mostram experimentos reais em pesquisa realizados nos laboratórios atualmente.

PCR Cycle for DNA Amplification

Description

Transcript

Tags