An MMLV reverse transcriptase mutant, its preparation method and application

By mutating specific amino acid sites and optimizing codons in M-MLV reverse transcriptase, a heat-resistant MMLV reverse transcriptase mutant was developed, solving the problem of reduced stability and activity of reverse transcriptase at high temperatures, and realizing efficient cDNA synthesis and rapid reverse transcription reaction.

CN119614534BActive Publication Date: 2026-03-13XIAN HUAWEI TECH CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2024-12-24
Publication Date
2026-03-13

AI Technical Summary

Technical Problem

Existing M-MLV reverse transcriptases suffer from reduced stability and activity at high temperatures, limiting their application in real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), especially when processing complex RNA templates, where they are inefficient.

Method used

By mutating specific amino acid sites of M-MLV reverse transcriptase, RNase H activity is reduced and thermal stability is improved, resulting in heat-resistant MMLV reverse transcriptase mutants, including sites such as E69K, L139P, T163K, and H204R. Codon optimization is then used to increase expression levels.

Benefits of technology

This technology enables efficient reverse transcription of complex RNA templates at 75°C, improving cDNA synthesis efficiency and quality, adapting to both ordinary and complex reverse transcription systems, shortening reverse transcription time, and enhancing thermal and storage stability.

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Abstract

This invention discloses an MMLV reverse transcriptase mutant, belonging to the field of biotechnology, obtained by designing mutations at specific sites of the wild-type MMLV reverse transcriptase. The mutation sites include combinations of one or more of the following: A32, S56, E69, L139, T163, T197, H204, S232, N249, T306, F309, T330, G424, L435, D449, D524, E562, D583, L603, H642, and T664. Compared with existing MMLV reverse transcriptases, the MMLV reverse transcriptase mutant of this invention exhibits higher reverse transcription temperature and rate. This invention also discloses a method for preparing the MMLV reverse transcriptase mutant and its application in reverse transcription reactions.
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Citation Information

Patent Citations

  • Thermal stability reverse transcriptase mutant and application thereof

    CN111647576A

  • Recombinant reverse transcriptase variants for improved performance

    US20240368567A1