描述Thermo Scientific Maxima H Minus Reverse Transcriptase (RT) was developed through in vitroevolution of M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity but lacks RNase H activity due to mutation in RNase H domain of M-MuLV RT. The engineered enzyme features dramatically improved thermostability, 50X higher processivity, robustness and increased synthesis rate compared to wild type M-MuLV RT.The eliminated RNase H activity enables the enzyme to produce very long RNA transcripts up to 20 kb. Due to its high thermostability, the enzyme maintains full activity during the entire reverse transcription reaction and generates high yields of cDNA. The reaction temperature can be increased up to 65°C for efficient transcription of RNA regions with a high secondary structure or to improve specificity using gene specific primers. The extremely high processivity of Maxima H Minus enzyme results in increased resistance to common reaction inhibitors, such as guanidine, formamide and ethanol. 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. |
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