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

- [Narrateur] Toutes les cellules du corps ont le m&#xEA;me ADN,mais ces cellules, comme les neurones et les cellulesmusculaires, expriment des g&#xE8;nes diff&#xE9;rents car seulscertains g&#xE8;nes sont transcrits en ARN messager, ou ARNmdans chaque cellule. 
En laboratoire, les ARNm peuvent servir de mod&#xE8;lepour synth&#xE9;tiser l&#x27;ADN compl&#xE9;mentaire, l&#x27;ADNc,pour &#xE9;tudier l&#x27;expression g&#xE9;n&#xE9;tique. 
Une m&#xE9;thode courante consiste &#xE0; extraire l&#x27;ARN des cellules,puis &#xE0; isoler l&#x27;ARNm d&#x27;autres types d&#x27;ARN,comme l&#x27;ARN ribosomal ou l&#x27;ARN de transfert,en faisant passer l&#x27;&#xE9;chantillon sur une colonne de billesavec des portions de thymines nucl&#xE9;otides fix&#xE9;s. 
Elles se lient &#xE0; la queue Poly(A),une cha&#xEE;ne de nucl&#xE9;otides ad&#xE9;nine sp&#xE9;cifiquement pr&#xE9;sentssur les trois premi&#xE8;res extr&#xE9;mit&#xE9;s de l&#x27;ARNm eucaryote. 
Les autres ARN ne se lient pas et sont &#xE9;limin&#xE9;s par lavage. 
Apr&#xE8;s l&#x27;isolement de l&#x27;ARNm, une amorce Poly(T)est li&#xE9;e &#xE0; la queue Poly(A), fournissant un point de d&#xE9;partaux enzymes de transcriptase inversepour transcrire un seul brin dans l&#x27;ADNc de l&#x27;ARNm. 
Des produits chimiques tels que les enzymes RNase sontensuite ajout&#xE9;s pour d&#xE9;grader l&#x27;ARN. 
Les enzymes ADN polym&#xE9;rase sont ensuite utilis&#xE9;espour synth&#xE9;tiser un brin compl&#xE9;mentaire de l&#x27;ADNc,ce qui donne un ADNc double brin,qui peut &#xEA;tre ins&#xE9;r&#xE9; dans un vecteur bact&#xE9;rien ou viralet utilis&#xE9; en biologie mol&#xE9;culaire.

Pray, Leslie A. « La révolution biotechnologique : PCR et l’utilisation de la transcriptase inverse pour cloner les gènes exprimés. » Nature Education 1, n° 1 (2008) : 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553 » target="__blank">Source</a>]

adn complémentaire

Complementary DNA

<h4>Aper&#xE7;u</h4>
 
 Seuls les g&#xE8;nes transcrits en ARN messager (ARNm) sont actifs ou exprim&#xE9;s. Les scientifiques peuvent donc extraire l&#x2019;ARNm des cellules pour &#xE9;tudier l&#x2019;expression des g&#xE8;nes dans diff&#xE9;rentes cellules et tissus. Le scientifique convertit l&#x2019;ARNm en ADN compl&#xE9;mentaire (ADNc) par transcription inverse. &#xC9;tant donn&#xE9; que l&#x2019;ARNm ne contient pas d&#x2019;introns (r&#xE9;gions non codantes) et autres s&#xE9;quences r&#xE9;gulatrices, l&#x2019;ADNc, contrairement &#xE0; l&#x2019;ADN g&#xE9;nomique, permet &#xE9;galement aux chercheurs de d&#xE9;terminer directement la s&#xE9;quence d&#x2019;acides amin&#xE9;s du peptide cod&#xE9; par le g&#xE8;ne. 
 
 <h4>La synth&#xE8;se de l&#x2019;ADNc</h4>
 
 L&#x2019;ADNc peut &#xEA;tre g&#xE9;n&#xE9;r&#xE9; par plusieurs m&#xE9;thodes, mais une mani&#xE8;re commune est d&#x2019;extraire d&#x2019;abord l&#x2019;ARN total des cellules, puis d&#x2019;isoler l&#x2019;ARNm des types plus pr&#xE9;dominants &#x2014; l&#x2019;ARN de transfert (ARNt) et l&#x2019;ARN ribosomal (ARNr). L&#x2019;ARNm eucaryote mature a une queue poly(A) &#x2014; une cha&#xEE;ne de nucl&#xE9;otides d&#x2019;ad&#xE9;nine &#x2014; ajout&#xE9;e &#xE0; son extr&#xE9;mit&#xE9; 3&#x2019;, alors que d&#x2019;autres types d&#x2019;ARN n&#x2019;en ont pas. Par cons&#xE9;quent, une cha&#xEE;ne de nucl&#xE9;otides de thymine (oligo-dTs) peut &#xEA;tre fix&#xE9;e &#xE0; un substrat tel qu&#x2019;une colonne ou des billes magn&#xE9;tiques, pour former un appariement de bases sp&#xE9;cifique avec les queues poly(A) de l&#x2019;ARNm. Alors que l&#x2019;ARNm avec une queue poly(A) est pi&#xE9;g&#xE9;, les autres types d&#x2019;ARN sont emport&#xE9;s lors du lavage. 
 
 Ensuite, la transcriptase inverse &#x2014; une enzyme d&#x2019;ADN polym&#xE9;rase des r&#xE9;trovirus &#x2014; est utilis&#xE9;e pour g&#xE9;n&#xE9;rer l&#x2019;ADNc &#xE0; partir de l&#x2019;ARNm. Puisque comme la plupart des ADN polym&#xE9;rases, la transcriptase inverse ne peut ajouter des nucl&#xE9;otides qu&#x2019;&#xE0; l&#x2019;extr&#xE9;mit&#xE9; 3&#x2019; d&#x2019;une cha&#xEE;ne, une amorce poly(T) est ajout&#xE9;e pour se lier &#xE0; la queue poly(A) afin de fournir un point de d&#xE9;part pour la synth&#xE8;se de l&#x2019;ADNc. Le brin d&#x2019;ADNc se termine en boucle d&#x2019;&#xE9;pingle &#xE0; cheveux. L&#x2019;ARN est alors d&#xE9;grad&#xE9; &#x2014; g&#xE9;n&#xE9;ralement avec un traitement alcalin ou des enzymes RNase &#x2014; laissant l&#x2019;ADNc &#xE0; simple brin intact.
 
 Un deuxi&#xE8;me brin d&#x2019;ADN compl&#xE9;mentaire &#xE0; l&#x2019;ADNc est ensuite synth&#xE9;tis&#xE9; par l&#x2019;ADN polym&#xE9;rase, souvent &#xE0; l&#x2019;aide de la boucle d&#x2019;&#xE9;pingle &#xE0; cheveux du premier brin d&#x2019;ADNc ou d&#x2019;un morceau entaill&#xE9; de l&#x2019;ARNm comme amorce. 
 
 L&#x2019;ADNc &#xE0; double brin qui en r&#xE9;sulte peut &#xEA;tre ins&#xE9;r&#xE9; dans des vecteurs bact&#xE9;riens ou viraux et clon&#xE9; &#xE0; l&#x2019;aide de techniques de biologie mol&#xE9;culaire standard. Une biblioth&#xE8;que d&#x2019;ADNc, repr&#xE9;sentant tous les ARNm dans les cellules ou les tissus d&#x2019;int&#xE9;r&#xEA;t, peut &#xE9;galement &#xEA;tre construite pour des recherches suppl&#xE9;mentaires.

ADN complémentaire

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

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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

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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

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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...

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Gene Delivery for Serious Diseases

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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

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La collection JoVE Core Biologie donne vie à la biologie à travers plus de 300 leçons vidéos animées concises et faciles à comprendre qui expliquent les concept clés de la biologie, mais aussi plus de 150 vidéos de scientifiques à l'action effectuant des expériences de recherche dans des laboratoires modernes.

From mRNA to cDNA for Gene Studies

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