4.7
ADN complementario
Descripción general
Sólo los genes que se transcriben en ARN mensajero (ARNm) están activos o se expresan. Por tanto, los científicos pueden extraer e…
- [Narrador] Casi todas las células 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 célula.
En el laboratorio, los ARNm son utilizados como un modelo
para sintetizar ADN complementario, o ADNc,
para estudiar la expresión genética.
Un método común es extraer ARN de las células,
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 nucleótidos de timina asociados.
Estos se unen al extremo poli-A,
una cadena de nucleótidos de adenina específicamente
presente en las terminaciones prima-3 de ARNm eucarionte.
Los otros ARN no se unen y son arrastrados con la muestra.
Después 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.
Químicos, como las enzimas ribonucleasas,
se añaden despúes 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 biología molecular.
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Q1: What is complementary DNA and how is it made?
Complementary DNA (cDNA) is synthesized from a messenger RNA (mRNA) template using the enzyme reverse transcriptase. This process converts the genetic information stored in RNA back into DNA form, creating a stable copy that represents only the expressed genes from a specific cell or tissue at a particular time.
Q2: Why is complementary DNA useful in molecular biology research?
Complementary DNA is valuable because it represents only actively expressed genes, eliminating introns and non-coding sequences found in genomic DNA. This makes cDNA ideal for studying gene expression patterns, cloning genes, and creating cDNA libraries that reflect the functional genes present in specific cell types or developmental stages.
Q3: How does complementary DNA differ from genomic DNA?
Complementary DNA contains only exons and represents genes actively transcribed in a cell, while genomic DNA includes all DNA sequences, including introns and regulatory regions. cDNA is synthesized from mRNA and lacks introns, making it smaller and more suitable for expressing eukaryotic genes in prokaryotic systems.
Q4: What role does reverse transcriptase play in cDNA synthesis?
Reverse transcriptase is the key enzyme that catalyzes cDNA synthesis by reading an mRNA template and synthesizing a complementary DNA strand. This enzyme enables researchers to convert the temporary RNA message into stable DNA that can be amplified, cloned, and analyzed using standard molecular biology techniques.
Q5: How can complementary DNA be amplified and analyzed?
Complementary DNA can be amplified using PCR or rapid amplification of cDNA ends (RACE) techniques, then analyzed through methods like agarose gel electrophoresis for separation of DNA fragments or sequencing. These approaches allow researchers to identify specific genes and determine their expression levels in different tissues or conditions.
Q6: What are cDNA libraries and why are they important?
A cDNA library is a collection of cloned cDNA sequences representing all genes expressed in a particular cell type or tissue. These libraries are important for functional genomics research, allowing scientists to study gene expression patterns, identify tissue-specific genes, and isolate genes for further characterization and protein production.
Q7: How is complementary DNA used in gene expression studies?
Complementary DNA serves as the template for measuring gene expression through techniques like real-time RT-PCR, which quantifies mRNA levels. Since cDNA represents only expressed genes, it provides an accurate snapshot of which genes are active in specific cells, tissues, or disease states.