ExcScript Reverse Transcriptase (RT) was developed through in vitro evolution of M-MuLV RT. The enzyme possesses RNA-dependent and DNA-dependent polymerase activity but lacks RNase H activity, which is due to a mutation in the RNase H domain of M-MuLV RT. Compared to wild type M-MuLV RT, the engineered enzyme has dramatically improved thermostability, inhibition resistance, 50-fold higher processivity, robustness, and increased synthesis rate. The lack of RNase H activity allows ExcScript to produce long RNA transcripts of up to 20 kb. Additionally, the enzyme's high thermostability enables it to maintain full activity during the entire reverse transcription reaction and generate high yields of cDNA. The reaction temperature can be increased up to 65°C to efficiently transcribe RNA regions with high secondary structures or to improve specificity using gene-specific primers. The high processivity of ExcScript results in increased resistance to common reaction inhibitors. Highlights • Thermostable—90% active after incubation at 50°C for 60 minutes in a reaction mixture• Active up to 65°C• High yields of full-length cDNA up to 20 kb• High sensitivity—reproducible cDNA synthesis from a wide range of starting total RNA amounts (1 pg to 5 µg)• Efficient—complete cDNA synthesis in 15 to 30 minutes• Increased resistance to common reaction inhibitors• Incorporates modified nucleotides Applications • First strand cDNA synthesis for RT-PCR and real-time RT-PCR.• Reverse transcription at elevated temperatures to reduce effects of secondary structure.• Synthesis of cDNA for cloning and expression.• Generation of labeled cDNA probes for microarrays.• Analysis of RNA by primer extension. Data Sheet: ExcScript Reverse Transcriptase
