4.7
DNA Complementar
Visão Geral
Apenas genes que são transcritos em RNA mensageiro (mRNA) estão ativos ou expressos. Os cientistas podem, portanto, extrair o mRNA das cél…
Quase todas as células do corpo tem o mesmo DNA, mas diferentes tipos de células, como neurônios e células musculares, expressam genes diferentes porque apenas alguns genes são transcritos em RNA mensageiro ou mRNA em cada célula. No laboratório, mRNAs podem ser usados como um modelo para sintetizar o DNA complementar, cDNA, para estudar a expressão do gene. Um método comum é extrair RNA das células, em seguida, isolar o mRNA de outros tipos de RNA, como RNA ribossómico ou RNA de transferência, executando a amostra sobre uma coluna de contas com trechos de nucleotídeos de timina anexados.
Estes se ligam à cauda poli uma cadeia de nucleotídeos de adenina especificamente presente nas três pontas principais do mRNA eucariótico. Os outros tipos de RNA não se ligam e são lavados. Após o mRNA ser isolado, um primer poli está ligado para a cauda poli fornecendo um ponto de partida para as enzimas de transcriptase reversa para transcrever uma fita simples cDNA do mRNA.
Químicos, como enzimas RNase são então adicionados para degradar o RNA. Enzimas de DNA polimerase são então 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.
View the full transcript and gain access to JoVE Core videos
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.