DNA and RNA are fundamental molecules that carry genetic information within living organisms. DNA typically exists as a double helix, while RNA is generally a single strand. Complementary DNA (cDNA) is a specific type of DNA whose structure often raises questions. This molecule plays a significant role in molecular biology.
The Initial Form: Single-Stranded cDNA
cDNA is initially synthesized as a single-stranded molecule. This occurs because it is created directly from a messenger RNA (mRNA) template. The process, known as reverse transcription, is carried out by an enzyme called reverse transcriptase.
Reverse transcriptase uses the single-stranded mRNA to assemble a complementary DNA strand. This enzyme binds to the RNA template, often using an oligo(dT) primer, and then synthesizes a DNA strand that mirrors the mRNA sequence. The newly formed DNA strand is therefore single-stranded.
Creating Double-Stranded cDNA
While cDNA starts as a single strand, it is frequently converted into a double-stranded form for various laboratory procedures. After the initial single-stranded cDNA is synthesized, the original mRNA template is removed. This removal can be facilitated by enzymes like RNase H.
The single-stranded cDNA then serves as a template for the synthesis of a second, complementary DNA strand. This process is catalyzed by DNA polymerase I. The result is a more stable double-stranded cDNA molecule. This double-stranded form is advantageous for applications requiring increased stability, such as gene cloning or amplification through Polymerase Chain Reaction (PCR).
Applications of cDNA
Scientists frequently utilize cDNA due to its advantages over genomic DNA. A primary benefit is that cDNA represents only the protein-coding regions, or exons, of a gene, as non-coding introns are removed during mRNA processing. This makes cDNA valuable for studying gene expression, as it directly reflects the genes actively being transcribed in a cell or tissue at a given time.
cDNA is also used in gene cloning, especially when expressing human genes in bacteria, which lack the machinery to process introns found in genomic DNA. By using cDNA, researchers ensure that only the relevant coding sequences are expressed. cDNA is also used to create gene libraries, which are collections of cloned cDNA fragments representing the mRNA content of a cell or tissue, providing a snapshot of expressed genes. This molecule also finds use in diagnostic applications, such as detecting RNA viruses by first converting their RNA genome into cDNA for subsequent analysis.