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

- [Narrador] Casi todas las
c&#xE9;lulas contienen el mismo ADN

pero algunas, como las neuronas
y las fibras musculares,

expresan genes diferentes

porque solo algunos genes son transcritos

en ARN mensajero, o ARNm, en cada c&#xE9;lula.

En el laboratorio, los ARNm
son utilizados como un modelo

para sintetizar ADN
complementario, o ADNc,

para estudiar la expresi&#xF3;n gen&#xE9;tica.

Un m&#xE9;todo com&#xFA;n es extraer
ARN de las c&#xE9;lulas,

luego aislar el ARNm de
los otros tipos de ARN,

como ARN ribosomal o ARN de transferencia,

dejando pasar la muestra
por una columna de cuentas

con segmentos de nucle&#xF3;tidos
de timina asociados.

Estos se unen al extremo poli-A,

una cadena de nucle&#xF3;tidos
de adenina espec&#xED;ficamente

presente en las terminaciones
prima-3 de ARNm eucarionte.

Los otros ARN no se unen y son
arrastrados con la muestra.

Despu&#xE9;s de aislar el ARNm,

un cebador poli-T se
une al extremo poli-A,

siendo un origen para que la
enzima transcriptasa inversa

transcriba una cadena sencilla
de ADNc a partir del ARNm.

Qu&#xED;micos, como las enzimas ribonucleasas,

se a&#xF1;aden desp&#xFA;es para degradar el ARN.

Entonces se utilizan
enzimas polimerasas de ADN

para sintetizar una cadena
complementaria para el ADNc,

obteniendo una cadena doble de ADNc,

el cual puede ser insertado
en un vector bacterial o viral

y utilizado en investigaciones
de biolog&#xED;a molecular.

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>]

adn complementario

Complementary DNA

<h4>Descripci&#xF3;n general</h4> S&#xF3;lo los genes que se transcriben en ARN mensajero (ARNm) est&#xE1;n activos o se expresan. Por tanto, los cient&#xED;ficos pueden extraer el ARNm de las c&#xE9;lulas para estudiar la expresi&#xF3;n gen&#xE9;tica en diferentes c&#xE9;lulas y tejidos. El cient&#xED;fico convierte el ARNm en ADN complementario (ADNc) mediante transcripci&#xF3;n inversa. Debido a que el ARNm no contiene intrones (regiones no codificantes) ni otras secuencias reguladoras, el ADNc, a diferencia del ADN gen&#xF3;mico, tambi&#xE9;n permite a los investigadores determinar directamente la secuencia de amino&#xE1;cidos del p&#xE9;ptido codificado por el gen. <h4>S&#xED;ntesis de ADNc</h4> El ADNc se puede generar mediante varios m&#xE9;todos, pero una forma com&#xFA;n es extraer primero el ARN total de las c&#xE9;lulas y luego aislar el ARNm de los tipos m&#xE1;s predominantes,ARN de transferencia (ARNt) y ribos&#xF3;mico (ARNr). El ARNm eucari&#xF3;tico maduro tiene una cola poli(A),una cadena de nucle&#xF3;tidos de adenina, agregada a su extremo 3&#x27;, aunque otros tipos de ARN no la tienen. Por lo tanto, se puede unir una cadena de nucle&#xF3;tidos de timina (oligo-dT) a un sustrato como una columna o perlas magn&#xE9;ticas, para emparejar espec&#xED;ficamente las bases con las colas poli(A) del ARNm. Mientras se captura el ARNm con una cola poli(A), los otros tipos de ARN se eliminan por lavado. A continuaci&#xF3;n, se utiliza la transcriptasa inversa,una enzima ADN polimerasa de los retrovirus,para generar ADNc a partir del ARNm. Dado que, como la mayor&#xED;a de las ADN polimerasas, la transcriptasa inversa puede agregar nucle&#xF3;tidos solo al extremo 3&#x27; de una cadena, se agrega un cebador poli(T) para unirse a la cola poli(A) y proporcionar un punto de partida para la s&#xED;ntesis de ADNc. La cadena de ADNc termina en un bucle en forma de horquilla. Luego, el ARN se degrada,com&#xFA;nmente con tratamiento alcalino o enzimas RNasa,dejando intacto el ADNc monocatenario. Luego, la ADN polimerasa sintetiza una segunda hebra de ADN complementaria al ADNc, a menudo utilizando el bucle en horquilla de la primera hebra de ADNc o un trozo mellado del ARNm como cebador. El ADNc bicatenario resultante puede insertarse en vectores bacterianos o virales y clonarse utilizando t&#xE9;cnicas de biolog&#xED;a molecular est&#xE1;ndar. Tambi&#xE9;n se puede construir una biblioteca de ADNc, que represente todos los ARNm de las c&#xE9;lulas o tejidos de inter&#xE9;s, para investigaciones adicionales.

ADN complementario

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.

From mRNA to cDNA for Gene Studies | JoVE K12

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From mRNA to cDNA for Gene Studies

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