Complementary DNA, or cDNA, is a fundamental tool in molecular biology. It is a synthetic form of DNA created in a laboratory setting using an RNA molecule as the starting template. The purpose of cDNA is to provide a stable, manageable copy of the genetic information actively being used by a cell, offering a snapshot of gene activity.
Why RNA Must Be Converted to DNA
A cell’s genetic blueprint is stored in genomic DNA (gDNA), which contains the complete set of instructions for the organism. In complex organisms, this gDNA is highly complicated and contains large sections of non-coding information. Genes within gDNA are structured with non-coding segments called introns, interspersed between coding segments called exons, which contain the instructions for building a protein.
Before a gene can be translated into a protein, the entire gene is first transcribed into a precursor RNA molecule. The non-coding introns are then physically cut out and discarded in a process called splicing. This leaves a mature messenger RNA (mRNA) molecule that consists only of the necessary coding information (exons). Scientists convert this processed mRNA into a DNA form to focus exclusively on the genes that are expressed and actively producing proteins.
Defining Complementary DNA
Complementary DNA is a double-stranded DNA molecule that is an exact, intron-free copy of a mature messenger RNA sequence. The “complementary” designation comes from the fact that its sequence is built to pair precisely with the RNA template used to create it. Unlike genomic DNA, which is massive and contains all regulatory and non-coding sequences, a cDNA molecule corresponds to a single, specific, already-processed gene transcript.
This difference in structure makes cDNA useful because it represents the gene in its most compact and functional form. The resulting cDNA sequence contains only the continuous, protein-coding sequence, making it significantly shorter than the genomic DNA sequence from which it originated. The stability of DNA compared to fragile RNA means the genetic information is preserved in a format easier to work with using standard molecular biology techniques.
How cDNA is Created
cDNA synthesis begins with the isolation and purification of messenger RNA (mRNA) from a biological sample. The process of converting this RNA back into a DNA molecule is known as reverse transcription. This specialized process relies on a unique enzyme called Reverse Transcriptase, which uses an RNA strand as a template to build a DNA strand.
The first step requires a short, single-stranded DNA molecule called a primer to bind to the mRNA template. An oligo(dT) primer is often used, which specifically attaches to the poly-A tail found at the end of most mature mRNA molecules. Once the primer is annealed, the Reverse Transcriptase enzyme synthesizes the first strand of DNA, creating a complementary DNA sequence. This initial step results in a hybrid molecule composed of one strand of RNA bound to one newly synthesized strand of cDNA.
To create the final, stable, double-stranded cDNA, the original RNA strand must be removed and replaced with a second strand of DNA. This is achieved using an enzyme called RNase H, which degrades the RNA component of the hybrid, along with other enzymes like DNA Polymerase. The DNA Polymerase then synthesizes the second strand of DNA, using the first cDNA strand as its template, resulting in a robust, double-stranded cDNA molecule.
Key Applications in Modern Biology
cDNA is a powerful resource for studying gene function and expression. One of its most frequent uses is in quantifying gene expression, where techniques like quantitative Polymerase Chain Reaction (qPCR) measure precisely how much of a specific gene’s mRNA was present in the original cell sample. Since the amount of mRNA correlates with the level of protein being produced, this allows researchers to determine which genes are active under specific conditions.
cDNA is also foundational for gene cloning and genetic engineering, especially when working with prokaryotic organisms like bacteria. Bacteria lack the cellular machinery to remove introns, so a complete eukaryotic gene would be non-functional if inserted directly from genomic DNA. By using the intron-free cDNA, scientists can insert a functional, protein-coding sequence into a bacterial vector, enabling the bacteria to produce large quantities of human or animal proteins, such as insulin. Furthermore, cDNA is used extensively in large-scale sequencing projects, where it provides the template for next-generation sequencing to map the entire set of actively expressed genes—known as the transcriptome—in a cell or tissue.