Home Applications DNA Amplification, PCR and qPCR RT-PCR & cDNA Synthesis cDNA Synthesis

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. WarmStart RTx Reverse Transcriptase is a unique, in silico designed RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits enzyme activity below 40°C. This enzyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template. RTx is a robust enzyme for RNA detection in amplification reactions and is particularly well suited for use in LAMP (loop-mediated isothermal amplification). The Template Switching RT Enzyme Mix features an RT that adds a few non-templated nucleotides after it reaches the 5′ end of the RNA template, enabling efficient template switching activity in a RT reaction. The Luna Reverse Transcriptase featured in Luna RT-qPCR/qPCR products possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C.

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Protocols for cDNA Synthesis
    Publications related to cDNA Synthesis
    • 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, 5340-9. PubMedID: 25740998, DOI: 10.1128/JVI.00064-15
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