There may be several thousand different proteins being produced in a cell at any one time, all of which have associated mRNA molecules. To identify any one of those mRNA molecules, the clones of each individual mRNA have to be synthesised. Libraries that represent the mRNA in a particular cell or tissue are termed cDNA libraries. mRNA cannot be used directly in cloning since it is too unstable. However, it is possible to synthesise complementary DNA molecules (cDNAs) to all the mRNAs from the selected tissue. The cDNA may be inserted into vectors and then cloned. The production of cDNA (complementary DNA) is carried out using an enzyme termed reverse transcriptase, which is isolated from RNA-containing retroviruses.
Reverse transcriptase is an RNA-dependent DNA polymerase, and will synthesise a first-strand DNA complementary to an mRNA template, using a mixture of the four dNTPs. There is also a requirement (as with all polymerase enzymes) for a short oligonucleotide primer to be present (Figure 1). With eukaryotic mRNA bearing a poly(A) tail, a complementary oligo(dT) primer may be used. Alternatively, random hexamers may be used that randomly anneal to the mRNAs in the complex. Such primers provide a free 3′-hydroxyl group that is used as the starting point for the reverse transcriptase. Regardless of the method used to prepare the first-strand cDNA, one absolute requirement is high-quality non-degraded mRNA; the integrity of the RNA should always be checked by gel electrophoresis. In yet another approach, a fraction of the extract may be used in a cell-free translation system, which, if intact mRNA is present, will direct the synthesis of proteins represented by the mRNA molecules in the sample.
Fig1. Strategies for producing fi rst-strand cDNA from mRNA.
Following the synthesis of the first DNA strand, a poly(dC) tail is added to its 3′ end, using terminal transferase and dCTP. This will also, incidentally, put a poly(dC) tail on the poly(A) of mRNA. Hydrolysis by alkali is then used to remove the RNA strand, leaving single-stranded DNA that can be used, like the mRNA, to direct the synthesis of a complementary DNA strand. The second-strand synthesis requires an oligo(dG) primer, base-paired with the poly(dC) tail, which is catalysed by the Klenow fragment of DNA pol I. The final product is double-stranded DNA, one of the strands being complementary to the mRNA. One further method of cDNA synthesis involves the use of RNase H. Here, the first-strand cDNA is produced as above with reverse transcriptase, but the resulting mRNA–cDNA hybrid is retained. RNase H is then used at low concentrations to nick the RNA strand. The resulting nicks expose 3′-hydroxyl groups that are used by DNA polymerase as a primer to replace the RNA with a second strand of cDNA (Figure 2).
Fig2. Second-strand cDNA synthesis using the RNase H method.
