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

- [Narratore] Quasi tutte
le cellule del corpo

hanno lo stesso DNA, ma diversi tipi

di cellule, come neuroni
e cellule muscolari,

esprimono geni diversi
perch&#xE9; solo determinati geni

sono trascritti in RNA messaggero,
o mRNA, in ogni cellula.

In laboratorio gli mRNA
sono utilizzati come modello

per sintetizzare l&#x27;DNA gratuito, cDNA,

per studiare l&#x27;espressione genica.

Un metodo comune &#xE8; quello di
estrarre l&#x27;RNA dalle cellule,

quindi isolare l&#x27;mRNA
da altri tipi di RNA,

come l&#x27;RNA ribosomiale o l&#x27;RNA transfer,

eseguendo il campione
su una colonna di perle

con tratti di nucleotidi
di timina attaccati.

Questi si legano alla coda Poly (A),

una catena di nucleotidi
adenina specificamente presente

sui tre estremi dell&#x27;mRNA eucariotico.

Gli altri tipi di RNA non si
legano e vengono lavati via.

Dopo che l&#x27;mRNA &#xE8; stato
isolato, un primer Poly (T)

&#xE8; legato alla coda Poly (A),
fornendo un punto di partenza

per gli enzimi della trascrittasi inversa

per trascrivere un singolo
filamento in cDNA dall&#x27;mRNA.

Vengono aggiunti prodotti
chimici come gli enzimi RNasi

per degradare l&#x27;RNA.

Vengono quindi utilizzati
enzimi DNA polimerasi

sintetizzare un filo
complementare al cDNA,

risultante in cDNA a doppio filamento,

che pu&#xF2; essere inserito in
un vettore batterico o virale

e utilizzato nella ricerca
di biologia molecolare.

Pray, Leslie A. "La rivoluzione biotecnologica: PCR e l'uso della trascrittasi inversa per clonare geni espressi". Educazione alla natura 1, n. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Sorgente</a>]

dna complementare

Complementary DNA

<h4>Panoramica</h4>

Solo i geni trascritti in RNA messaggero (mRNA) sono attivi o espressi. Gli scienziati possono, quindi, estrarre l&#x27;mRNA dalle cellule per studiare l&#x27;espressione genica in diverse cellule e tessuti. Lo scienziato converte l&#x27;mRNA in DNA complementare (cDNA) attraverso la trascrizione inversa. Poich&#xE9; l&#x27;mRNA non contiene introni (regioni non codificanti) e altre sequenze regolatorie, il cDNA, a differenza del DNA genomico, consente anche ai ricercatori di determinare direttamente la sequenza di amminoacidi del peptide codificato dal gene.

<h4>sintesi cDNA</h4>

il cDNA pu&#xF2; essere generato con diversi metodi, ma un modo comune &#xE8; quello di estrarre prima l&#x27;RNA totale dalle cellule, e quindi isolare l&#x27;mRNA dai tipi pi&#xF9; predominanti: RNA di trasferimento (tRNA) e ribosomale (rRNA). L&#x27;mRNA eucariotico maturo ha una coda poli(A), una stringa di nucleotidi di adenina, aggiunta alla sua estremit&#xE0; 3&#x27;, mentre altri tipi di RNA no. Pertanto, una stringa di nucleotidi di timina (oligo-dT) pu&#xF2; essere attaccata a un substrato come una colonna o perline magnetiche, in modo specifico coppia di base con le code poli(A) di mRNA. Mentre l&#x27;mRNA con una coda poli(A) viene catturata, gli altri tipi di RNA vengono lavati via.

Successivamente, la trascrizione inversa, un enzima di polimerasi del DNA proveniente da retrovirus, viene utilizzata per generare cDNA dall&#x27;mRNA. Poich&#xE9;, come la maggior parte delle polimerasi del DNA, la trascrizione inversa pu&#xF2; aggiungere nucleotidi solo all&#x27;estremit&#xE0; 3&#x27; di una catena, viene aggiunto un primer poli(T) da associare alla coda poli(A) per fornire un punto di partenza per la sintesi cDNA. Il filocila cDNA termina in un ciclo di tornante. L&#x27;RNA viene quindi degradato, comunemente con il trattamento con alcali o enzimi RNAsi, lasciando intatto il cDNA a singolo filamento.

Un secondo filamento di DNA complementare al cDNA viene quindi sintetizzato dalla polimerasi del DNA, spesso usando il ciclo del primo filamento cDNA o un pezzo nicked dell&#x27;mRNA come primer.

Il cDNA a doppio filamento risultante pu&#xF2; essere inserito in vettori batterici o virali e clonati utilizzando tecniche di biologia molecolare standard. Una libreria cDNA, che rappresenta tutti gli mRNA nelle cellule o nel tessuto di interesse, pu&#xF2; anche essere costruita per ulteriori ricerche.

DNA Complementare

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

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 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

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

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From mRNA to cDNA for Gene Studies

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