To create a cDNA library, cDNA can be inserted into vectors and cloned. In the analysis of cDNA clones, it is often difficult to find clones that are full length—that is, many clones are shorter than the mature mRNA molecules from which they are derived. Why is this so?

Key Concepts

cDNA Synthesis

cDNA, or complementary DNA, is synthesized from mRNA through the action of the enzyme reverse transcriptase. This process involves the reverse transcription of mRNA into a DNA strand, which can then be amplified and cloned. However, during this synthesis, incomplete reverse transcription can occur, leading to shorter cDNA fragments that do not represent the full length of the original mRNA.

Introns and Exons

Eukaryotic genes are composed of exons (coding regions) and introns (non-coding regions). During mRNA processing, introns are removed, and exons are spliced together to form mature mRNA. When creating a cDNA library, if the cDNA is derived from pre-mRNA or if splicing errors occur, the resulting cDNA may lack some exons, resulting in shorter clones that do not fully represent the mature mRNA.

Cloning Efficiency

The efficiency of cloning cDNA into vectors can vary based on several factors, including the quality of the cDNA, the vector system used, and the transformation process. Some cDNA fragments may be preferentially amplified or selected during cloning, leading to a bias towards shorter clones. This can make it challenging to isolate full-length cDNA clones, as they may be outcompeted by shorter, more easily cloned fragments.