In the standard Gubler and Hoffman method for double-stranded cDNA synthesis, the first strand is synthesized from mRNA by reverse transcriptase (such as AMV-RT or M-MuLV RT). RNase H specifically cleaves the phosphodiester bonds of the RNA strand in an RNA:DNA hybrid, generating small RNA fragments. These residual RNA fragments act as primers for E. coli DNA polymerase I to synthesize the second cDNA strand via nick-translation, after which DNA ligase seals any remaining nicks.