What is the workflow of cDNA library construction?
Posted March 1, 2023
- mRNA is isolated - A cDNA library is constructed with mRNA, which carries the encoded information from DNA to ribosomes to be translated into protein. To start the construction of the cDNA library, the total mRNA is first isolated from a cell type or tissue of interest. Removing highly abundant tRNAs and rRNAs may be desirable, as they may make up a major percentage of the final library, making it difficult to detect low abundance RNAs.
- The first cDNA strand is synthesized - mRNA is not a substrate for DNA ligase and it cannot be cloned because it is single stranded. Before inserting the mRNA into a suitable vector, it is first converted into DNA. Primer is not required. The 3´end of the single-stranded cDNA acts as its own primer generating a short hairpin loop at this site.
- The second cDNA strand is synthesized - Either RTase or E.coli DNA polymerase is used to convert single stranded cDNA into double stranded (ds) cDNA. Using the minimum number of amplification cycles necessary to obtain sufficient material for sequencing is important. This prevents over-amplification of the library, which is the most common source of bias in the results.
- cDNA is incorporated into a vector - A blunt–ended double stranded cDNA molecule is obtained by using S1 nuclease to trim the ds cDNA. This is followed by addition of terminal transferase to tail the cDNA with C's and ligation into a vector. Short restriction-site linkers are first ligated to both ends to boost the efficiency of blunt-end ligation.
- cDNAs are cloned - cDNAs are typically cloned in phage insertion vectors. Bacteriophage vectors, which are capable of handling and storing larger numbers of phage clones, are preferred over plasmid vectors.