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Complementary DNA
Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study ge…
Almost every cell in the body has the same DNA.
However, different cell types, such as neurons and muscle cells, express different sets of genes. These genes are transcribed into different messenger RNAs, or mRNAs, in each cell.
In the laboratory, mRNA can be used as a template to synthesize complementary DNA, or cDNA. Since cDNA is synthesized from cellular mRNA, it represents the genes expressed by the cell.
One common method begins with extracting RNA from cells.
Following cell lysis and removal of cellular debris, the mRNA is isolated from other types of RNA, such as ribosomal RNA and transfer RNA. This is commonly done by adding the sample to a column containing beads with short stretches of thymine nucleotides attached.
These thymine stretches bind to the poly-A tail, a chain of adenine nucleotides found at the 3-prime end of eukaryotic mRNA, through complementary base pairing.
Other types of RNA lack poly-A tails, so they do not bind to the column and are washed away.
An elution buffer with a low salt concentration is then added. This disrupts the interaction between the poly-A tail and the thymine stretches, releasing the bound mRNA from the beads.
After the mRNA is isolated, an oligo-dT primer binds to the poly-A tail and provides a starting point for reverse transcriptase enzymes to synthesize a single-stranded cDNA copy of the mRNA.
RNase enzymes are then added to degrade the RNA strand of the RNA-cDNA hybrid.
Next, DNA polymerase enzymes synthesize a complementary DNA strand, producing double-stranded cDNA.
This cDNA can then be inserted into plasmids or viral vectors for molecular biology research.
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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.