4.7
互补DNA
“只有转录成信使RNA（mRNA）的基因才是活跃的或表达的。因此，科学家可以从细胞中提取mRNA来研究不同细胞和组织中的基因表达。科学家通过反转录将mRNA转化为互补DNA（cDNA）。由于mRNA不包含内含子（非编码区）和其它调控序列，cDNA与基因组DNA不同，它还允许研究人员直接确定由基因编码…
Almost every cell in the body has the same DNA, but different cell types, such as neurons and muscle cells, express different genes because only certain genes are transcribed into messenger RNA, or mRNA, in each cell. In the laboratory, mRNAs can be used as a template to synthesize complementary DNA, cDNA, to study gene expression. A common method is to extract RNA from cells, then isolate the mRNA from other types of RNA, like ribosomal RNA or transfer RNA, by running the sample over a column of beads with stretches of thymine nucleotides attached.
These bind to the poly-A tail, a chain of adenine nucleotides specifically present on the 3-prime ends of eukaryotic mRNA. The other types of RNA do not bind and are washed away.
After the mRNA is isolated, a poly-T primer is bound to the poly-A tail, providing a starting point for reverse transcriptase enzymes to transcribe a single-stranded cDNA from the mRNA. Chemicals, such as RNase enzymes, are then added to degrade the RNA.
DNA polymerase enzymes are then used to synthesize a strand complementary to the cDNA, resulting in double-stranded cDNA, which can be inserted into a bacterial or viral vector and used in molecular biology research.
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.