A cDNA library is a collection of clones containing cDNAs. cDNA libraries are often intended to represent as many as possible of the mRNAs contained within a cell. Because working with mRNA is difficult (as mRNA is unstable and is easily degraded by RNases which can be found even on the skin), researchers use an enzyme called reverse transcriptase which will produce a DNA copy of each mRNA strand. Referred to as cDNA these reverse transcribed mRNAs are collectively known as the library.
Such a library has several uses. A cDNA of an eukaryotic organism (for example, a human) can be cloned into a prokaryotic organism (for example, E. coli) and expressed (translated into the appropriate protein) there (with limitations, for example posttranslational modification). A cDNA library is also important for analysis through bioinformatics. The complete cDNA library of an organism gives the total of the proteins it can possibly express. Also, comparison of cDNA sequence between libraries constructed from cells derived from different organisms can provide insight into the genetic and evolutionary relationship between organisms through the similarity of their cDNA.
cDNA Library Construction
cDNA libraries are created for the purpose of cataloging sections of mRNA. Hence, the mRNA to be catalogued must first be isolated from the cell and purified through various means. Once the purified mRNA is obtained, reverse transcriptase is used to make templates of DNA from the mRNA templates. The newly formed DNA templates are then inserted into bacteria plasmids using restriction enzymes (to cut open the plasmid) and ligase (to seal up the ends once the DNA has annealed into the plasmid).
Unfortunately, a very low percentage of plasmids actually have the DNA successfully inserted into the plasmid despite a high percentage cut by restriction enzymes. Many plasmids simply remain cut open or anneal with itself again. Therefore, to isolate those plasmids that do have the DNA successfully inserted, an extra gene is usually inserted along with the DNA to confer antibiotic resistance.
The plasmids are then reinserted into bacteria to grow in a medium with this antibiotic. All bacteria with plasmids that have successfully taken in the DNA (and hence the antibiotic resistance), will then survive, with all the others dying off. The surviving colonies are then grown to reproduce the plasmid with the DNA, which is then purified. This is also why these types of libraries are called cDNA or complementary DNA, because it is a complemetary strand of DNA to the mRNA in question.
cDNA Library vs. Genomic DNA Library
In Eukaryotic genomes, the entire sequence of genes is often very large because coding sequence (exons) is interrupted by introns that do not contain coding DNA sequence. The advantage of a cDNA library is that only the exons, or DNA representing expressed sequence, are templates for the creation of DNA (via Reverse Transcriptase) to collect the preferred genes. On the other hand, genes from prokaryotic cells does not usually contain introns, and the DNA is therefore much shorter. Also, prokaryotic mRNA can be difficult to isolate; because of this a genomic library is more preferable for prokaryotic genomic studies.
- cDNA LIBRARY- Properties, Construction, Isolation & Techniques available for cDNA cloning
- Functional Annotation of the Mouse database
- examples of cDNA synthesis and cloning