M-MLV RT mutant and its polynucleotides, preparation methods, enzyme activity detection methods and RNA reverse transcription methods, vectors containing this mutant, recombinant engineered cell lines and reagent kits.

Abstract

The application provides a novel high-temperature-resistant and inhibitor-resistant M-MLV RT mutant, a polynucleotide molecule, a preparation method, an enzyme activity detection method, an RNA reverse transcription method, a vector containing the M-MLV RT mutant, a recombinant engineering cell strain and a kit. Specifically, the application establishes an RNA aptamer-based method for detecting M-MLV RT enzyme activity, and through site-directed mutagenesis and combined mutation, finally screens out a high-temperature-resistant M-MLV RT mutant (Max5.0), and the amino acid sequence is shown as SEQ ID NO. 8. Compared with natural M-MLV RT and commercial SuperScript III reverse transcriptase, the M-MLV RT mutant can still perform efficient reverse transcription at 65 DEG C, has good resistance to KCl and formamide, and has high reaction sensitivity.

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