逆转录酶
To prepare a cDNA library, the first step is to isolate the total mRNA from the cell type of interest. Then, the enzyme reverse transcriptase is used to generate cDNAs. Reverse transcriptase is a RNA-dependent DNA polymerase. It depends on the presence of a primer, usually a poly-dT oligonucleotide, to prime DNA synthesis. DNA polymerase can use these single–stranded primers to initiate second strand DNA synthesis on the mRNA templates. After the single-stranded DNA molecules are converted into double-stranded DNA molecules by DNA polymerase, they are inserted into vectors and cloned. To do this, the cDNA are frequently methylated with a specific methyl transferase. These synthetic oligonucleotides can either be “linkers”, the overhangs are generated by digestion with the appropriate restriction enzyme.