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.
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Gubler, U. Second-strand cDNA synthesis: mRNA fragments as primers. Methods Enzymol. 152, 330–335 (1987).
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Synthesis of complementary DNA. Nat Methods 2, 151–152 (2005). https://doi.org/10.1038/nmeth0205-151
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DOI: https://doi.org/10.1038/nmeth0205-151
