Name: from mrna to cdna for gene studies
Uploaded: 2026-08-26T08:20:09.925Z
Duration: 2 min 25 s
Channel: JoVE

Quase todas as c&#xE9;lulas do corpo tem o mesmo DNA, mas diferentes tipos de c&#xE9;lulas, como neur&#xF4;nios e c&#xE9;lulas musculares, expressam genes diferentes porque apenas alguns genes s&#xE3;o transcritos em RNA mensageiro ou mRNA em cada c&#xE9;lula. No laborat&#xF3;rio, mRNAs podem ser usados como um modelo para sintetizar o DNA complementar, cDNA, para estudar a express&#xE3;o do gene. Um m&#xE9;todo comum &#xE9; extrair RNA das c&#xE9;lulas, em seguida, isolar o mRNA de outros tipos de RNA, como RNA riboss&#xF3;mico ou RNA de transfer&#xEA;ncia, executando a amostra sobre uma coluna de contas com trechos de nucleot&#xED;deos de timina anexados.Estes se ligam &#xE0; cauda poli uma cadeia de nucleot&#xED;deos de adenina especificamente presente nas tr&#xEA;s pontas principais do mRNA eucari&#xF3;tico. Os outros tipos de RNA n&#xE3;o se ligam e s&#xE3;o lavados. Ap&#xF3;s o mRNA ser isolado, um primer poli est&#xE1; ligado para a cauda poli fornecendo um ponto de partida para as enzimas de transcriptase reversa para transcrever uma fita simples cDNA do mRNA.Qu&#xED;micos, como enzimas RNase s&#xE3;o ent&#xE3;o adicionados para degradar o RNA. Enzimas de DNA polimerase s&#xE3;o ent&#xE3;o utilizadas para sintetizar um fio complementar ao cDNA resultando em cDNA de fita dupla, que pode ser inserido em um vetor bacteriano ou viral e usado em pesquisas de biologia molecular.

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>]

dna complementar

Complementary DNA

<h4>Vis&#xE3;o Geral</h4>
 
 Apenas genes que s&#xE3;o transcritos em RNA mensageiro (mRNA) est&#xE3;o ativos ou expressos. Os cientistas podem, portanto, extrair o mRNA das c&#xE9;lulas para estudar a express&#xE3;o gen&#xE9;tica em diferentes c&#xE9;lulas e tecidos. O cientista converte o mRNA em DNA complementar (cDNA) atrav&#xE9;s de transcri&#xE7;&#xE3;o reversa. Como o mRNA n&#xE3;o cont&#xE9;m intr&#xF5;es (regi&#xF5;es n&#xE3;o codificantes) e outras sequ&#xEA;ncias regulat&#xF3;rias, o cDNA&#x2014;ao contr&#xE1;rio do DNA gen&#xF3;mico&#x2014;tamb&#xE9;m permite que os investigadores determinem diretamente a sequ&#xEA;ncia de amino&#xE1;cidos do pept&#xED;deo codificado pelo gene.
 
 <h4>S&#xED;ntese de cDNA</h4>
 
 O cDNA pode ser gerado por v&#xE1;rios m&#xE9;todos, mas uma maneira comum &#xE9; primeiro extrair o RNA total das c&#xE9;lulas e, em seguida, isolar o mRNA dos tipos mais predominantes&#x2014;RNA de transfer&#xEA;ncia (tRNA) e riboss&#xF3;mico (rRNA). O mRNA eucari&#xF3;tico maduro tem uma cauda poli(A)&#x2014;uma sequ&#xEA;ncia de nucle&#xF3;tidos de adenina&#x2014;adicionada ao seu terminal 3&#x2019;, enquanto que outros tipos de RNA n&#xE3;o. Portanto, uma cadeia de nucle&#xF3;tidos de timina (oligo-dTs) pode ser anexada a um substrato, como uma coluna ou contas magn&#xE9;ticas, para emparelhar as bases especificamente com as caudas poli(A) do mRNA. Enquanto que o mRNA com cauda poli(A) &#xE9; capturado, os outros tipos de RNA s&#xE3;o lavados.
 
 Em seguida, a transcriptase reversa&#x2014;uma enzima de DNA polimerase de retrov&#xED;rus&#x2014;&#xE9; usada para gerar cDNA a partir do mRNA. Uma vez que, como a maioria das DNA polimerases, a transcriptase reversa pode apenas adicionar nucle&#xF3;tidos ao terminal 3&#x2019; de uma cadeia, um primer poli(T) &#xE9; adicionado para se ligar &#xE0; cauda poli(A) para fornecer um ponto de partida para a s&#xED;ntese de cDNA. A cadeia de cDNA termina em um hairpin loop. O RNA &#xE9; ent&#xE3;o degradado&#x2014;comumente com tratamento alcalino ou enzimas RNase&#x2014;deixando o cDNA de cadeia simples intacto.
 
 Uma segunda cadeia de DNA complementar ao cDNA &#xE9; ent&#xE3;o sintetizada pela DNA polimerase&#x2014;muitas vezes usando o hairpin loop da primeira cadeia do cDNA ou uma por&#xE7;&#xE3;o cortada do mRNA como primer.
 
 O cDNA de cadeia dupla resultante pode ser inserido em vectores bacterianos ou virais e clonado usando t&#xE9;cnicas padr&#xE3;o de biologia molecular. Uma biblioteca de cDNA&#x2014;representando todos os mRNAs nas c&#xE9;lulas ou tecidos de interesse&#x2014;tamb&#xE9;m pode ser criada para investiga&#xE7;&#xF5;es adicionais.

DNA Complementar

Overview

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to ...

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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 researchers determine the amino acid sequence of the peptide encoded by a gene, unlike genomic DNA.

To make cDNA, scientists often start by extracting total RNA from cells. They then isolate mRNA from the more abundant transfer RNA (tRNA) and ribosomal RNA (rRNA). Mature eukaryotic mRNA has a poly(A) tail, which is a chain of adenine nucleotides added to the 3' end. Other RNA types do not have this feature.

Because of the poly(A) tail, oligo-dT molecules can be attached to a column or magnetic beads to capture mRNA by base pairing. The other RNA molecules are washed away. Next, reverse transcriptase, a DNA polymerase enzyme from retroviruses, builds cDNA from the mRNA template. A poly(T) primer binds to the poly(A) tail and gives the enzyme a starting point, since it adds nucleotides only to the 3' end of a chain.

The first cDNA strand ends in a hairpin loop. The RNA is then degraded, often with alkali treatment or RNase enzymes, and the single-stranded cDNA remains. A second DNA strand is then synthesized by DNA polymerase. It may use the hairpin loop of the first strand or a nicked piece of mRNA as a primer. The final double-stranded cDNA can be inserted into bacterial or viral vectors and cloned with standard molecular biology methods. Researchers can also build a cDNA library that represents the mRNAs found in a specific cell or tissue sample.

From mRNA to cDNA for Gene Studies

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

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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 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...

complementary dna

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

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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 scientists isolate mRNA, use reverse transcription, and make cDNA for gene expression studies, cloning, and cDNA libraries in cells and tissues.

From mRNA to cDNA for Gene Studies | 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.

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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.

From mRNA to cDNA for Gene Studies

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