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cDNA Synthesis

cDNA Synthesis describes the generation of complementary DNA (cDNA) from an RNA template by reverse transcription. Reverse transcriptases (RTs) use an RNA template and a primer complementary to the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.

Many RTs are available from commercial suppliers. Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. M-MuLV Reverse Transcriptase lacks 3´ → 5´ exonuclease activity. ProtoScript® II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.

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Protocols for cDNA Synthesis

    Publications related to cDNA Synthesis

  1. Terry Fei Fan Ng, Nikola O Kondov, Xutao Deng, Alison Van Eenennaam, Holly L Neibergs, Eric Delwart 2015. A metagenomics and case-control study to identify viruses associated with bovine respiratory disease. J Virol. 89, PubMedID: 25740998, DOI: 10.1128/JVI.00064-15

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