cDNA

cDNA is the acronym for "complementary DNA". This is an artificially produced DNA that is created in a molecular biology laboratory from mRNA(messenger RNA). cDNA is therefore the DNA copy of an active gene. It is produced from mRNA (messenger RNA), contains only exons, is stable and is constantly used in research, diagnostics and molecular biology.

The reverse transcription of RNA, coupled with the amplification of the resulting cDNA using polymerase chain reaction (RT-PCR), is one of the most important molecular biology technologies of the present day and is used in all areas of science and medicine (Bustin SA et al. 2022).

The technical process:

1. In a cell, a gene is first transcribed into an mRNA.
2. This mRNA contains only the genetic information of the expressed gene (i.e. without introns).
3. In a further step, the mRNA is "translated back" into DNA using the enzyme "reverse transcriptase".
4. The result is a single-stranded DNA - the cDNA.
5. In most cases, a double-stranded cDNA is then produced from this.

Important: mRNA is the molecule in a cell that indicates which gene is active at the time of examination (the extent of activity can also be determined).

Some important details distinguish a cDNA from genomic (normal) DNA. In particular, it lacks introns (non-coding sections). Introns have already been cut out of the mRNA. Since the cDNA is a mirror image of the mRNA, the cDNA also contains only the coding regions (exons) of a gene. This is one of the main technical reasons why it is easier to clone a gene without introns or to measure its activity.

A cDNA is mainly used as follows:

• Gene expression analysis: Methodologically, for example, the qPCR detection method is used, which analyzes in real time and fluorescence-based (RT-qPCR) which genes are active and how the strength of their activity can be measured. Gene expression analysis has long been used as a core technology for the precise, rapid and sensitive laboratory diagnosis of infectious diseases or in tumor and mutation diagnostics (Bustin SA et al. 2022). The key result of a qPCR is the Ct value (cylce threshold), which provides information on the number of PCR cycles after which the fluorescence exceeds the detection limit (low value = extensive starting material).
• Preparation of cDNA libraries. The production of full-length cDNA libraries enables researchers to study gene expression and protein interactions and to discover new genes. The techniques now allow the production of high-quality cDNA libraries with small amounts of mRNA. Another important step in the creation of high-quality cDNA libraries is "normalization". The differences in gene expression can be several orders of magnitude. Therefore, normalization of the cDNA library is essential. Using the enzyme "duplex-specific nuclease", it is possible to normalize the cDNA library based on the fact that more abundant molecules are more likely to reassemble after denaturation than rare molecules (Kooiker M et al. 2014).
• Cloning of genes, especially eukaryotic genes without introns
• RNA sequencing evaluation
• Diagnostic tests where only the expressed genes are of interest.

1. Bogdanova EA et al. (2008) Normalization of full-length enriched cDNA. Mol Biosyst 4: 205-212.
2. Bustin SA et al. (2022) RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step. Int J Mol Sci 23:1303.
3. Kooiker M et al (2014) cDNA library preparation. Methods Mol Biol1099:29-40.