Complementary DNA, or cDNA, is a synthetic form of DNA generated from a messenger RNA (mRNA) template. The “c” in its name signifies “complementary,” as it is created as a matching copy of the mRNA. This process captures which genes are actively being expressed within a cell at a specific moment, allowing researchers to isolate and study the coding sequences that hold the instructions for building proteins.
Creating cDNA from RNA
The creation of cDNA from RNA is called reverse transcription, a reaction that reverses the typical flow of genetic information from DNA to RNA. This process is guided by an enzyme called reverse transcriptase, which was first discovered in retroviruses like HIV. These viruses use the enzyme to convert their RNA genome into DNA after entering a host cell.
To create cDNA in a laboratory, scientists first isolate all the messenger RNA (mRNA) from a cell or tissue sample. Once isolated, a short DNA sequence called a primer is added. An “oligo-dT” primer is often used, which attaches to the poly-A tail found on most eukaryotic mRNA molecules to ensure only mRNA is targeted.
With the primer attached, reverse transcriptase is introduced to read the mRNA sequence and synthesize a single, complementary strand of DNA. After this synthesis, the original mRNA strand is removed. Other enzymes then create a second DNA strand, resulting in a stable, double-stranded cDNA molecule ready for various applications.
Key Differences Between cDNA and Genomic DNA
A cell’s genomic DNA (gDNA) is its complete genetic blueprint, containing every gene an organism possesses along with vast non-coding regions. In contrast, cDNA only includes the genes that were actively being transcribed into mRNA in the source tissue. This makes cDNA a direct reflection of cellular activity instead of a static library of all possible genetic instructions.
cDNA derived from eukaryotes—organisms with a cell nucleus—differs structurally from gDNA. Eukaryotic genes contain both exons (coding sequences) and introns (non-coding sequences). During the natural process of creating mRNA, these introns are spliced out. Since cDNA is synthesized from this mature mRNA, it contains only the protein-coding exon sequences.
The absence of introns makes cDNA molecules shorter and less complex than their corresponding regions in genomic DNA. For example, a gene in gDNA might be thousands of base pairs long due to large introns, while its cDNA copy may only be a few hundred. This compact nature, and the fact that DNA is more chemically stable than RNA, makes cDNA easier to handle, store, and manipulate in laboratory settings.
How Scientists Use cDNA
Scientists can measure the amount of a specific cDNA to determine how active a particular gene is within a cell. Techniques like Reverse Transcription Polymerase Chain Reaction (RT-PCR) and quantitative PCR (RT-qPCR) use cDNA to detect and quantify RNA levels with high sensitivity. These methods reveal how gene expression changes in response to different conditions or over time.
For a broader view, researchers employ technologies like DNA microarrays and RNA-Sequencing (RNA-Seq). A microarray involves placing thousands of known cDNA sequences on a slide to see which ones bind to labeled cDNA from a sample, providing a snapshot of many genes at once. RNA-Seq is a modern approach that sequences all the cDNA in a sample, offering a comprehensive profile of the entire transcriptome, or the full range of expressed genes.
cDNA is also used for gene cloning and producing proteins. Because cDNA lacks introns, it can be inserted into simple organisms like bacteria or yeast, which cannot splice them out. This allows scientists to use these host cells as “factories” to produce large quantities of a specific protein for study or therapeutic use.
Scientists also compile cDNA libraries, which are collections of all the cDNA from a particular tissue. These libraries create a lasting resource for discovering and isolating new genes.
cDNA’s Role in Medicine and Biotechnology
cDNA is used for diagnostics, especially for detecting RNA viruses. Pathogens such as influenza, HIV, and SARS-CoV-2 have RNA genomes. To detect these viruses, patient samples are treated with reverse transcriptase to convert any viral RNA into cDNA, which can then be amplified and identified using PCR-based tests for an accurate diagnosis.
In oncology and the diagnosis of genetic disorders, creating cDNA from a patient’s tissue, such as a tumor, allows clinicians to analyze gene expression patterns. These patterns can reveal which genes are overactive or inactive, providing insights into the type of cancer and its aggressiveness. This approach helps personalize medicine by tailoring therapies to the specific molecular characteristics of a disease.
The development of therapeutic drugs uses cDNA. Many modern medicines, including insulin, growth factors, and monoclonal antibodies, are proteins. To manufacture these biopharmaceuticals, the cDNA encoding the desired protein is inserted into host cells, which then produce the therapeutic protein in large quantities. This method of creating recombinant proteins has advanced the treatment of numerous conditions, from diabetes to autoimmune diseases.