cDNA Library

Written by Super User. Posted in DNA

Learn about cDNA libraries.

Learn about cDNA libraries.

cDNA Libraries

cDNA libraries or complementary DNA libraries are a collection of cloned cDNA fragments. cDNA libraries are powerful and useful tools as they are able to contain all the coding information (the genetic code that produces proteins) from a given cell population.

 

cDNA Library Generation

cDNA libraries diagram of how to generate them

cDNA Synthesis Picture

cDNA library

RNA needs to first be isolated from a cell population. Several methods are commonly used in order to purify RNA, these include kit based polyA column methods and other chemical methods such as trizol extraction. A poly-(A) tail (consisting of a long repeating sequence of adenine nucleotides) is used in eukaryotic cells as a primer site for reverse transcription. cDNA is created from mature mRNA using the reverse transcriptase enzyme which generates complementary DNA from the RNA. This also allows the specific priming from mRNAs and not other RNAs as mRNAs contain a poly-A tail at their 3' end. Once the mRNA is isolated, oligo-dT or random primers can be used with reverse transcriptase to create cDNA templates. Restriction endonucleases and DNA ligase are then used to clone the cDNA sequences into bacterial plasmids.

After the cDNA is synthesized, it is cloned into expression vectors or plasmids. These plasmids each containing one cDNA are transformed into bacterial competent cells (made competent by a variety of chemical or other methods in order to allow them to take up the plasmid with the cDNA). These plasmids are then amplified in the growing bacteria. The bacteria clones are then selected so that only bacteria containing the plasmid will survive. This is commonly done through antibiotic resistance selection.

Once the bacteria are selected, stocks of the bacteria are created which can later be grown and sequenced to compile a cDNA library. display_block('cdna'); ?>