The basic strategy for enzymatic conversion of RNA into DNA has changed little since the 1970s; however, there have been great improvements to the efficiency of the overall process. In this method1,2,3, the product of a first-strand synthesis (the cDNA-mRNA hybrid) is used as template for a nick translation reaction. Ribonuclease (RNase) H produces nicks and gaps, creating a series of RNA primers used by Escherichia coli DNA polymerase I during the synthesis of the second-strand DNA. Residual nicks are then repaired by E. coli DNA ligase, and the frayed termini of the double-stranded cDNA are polished by a DNA polymerase. Finally, bacteriophage T4 polynucleotide kinase catalyzes the phosphorylation of the ends of the cDNAs to facilitate cloning.
