期刊
NUCLEIC ACIDS RESEARCH
卷 37, 期 2, 页码 473-481出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkn952
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资金
- Agilent Technologies
In an effort to increase the thermostability of Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), we screened random and site-saturation libraries for variants that show increased resistance to thermal inactivation. We discovered five mutations E69K, E302R, W313F, L435G and N454K that collectively increase the half-life of MMLV RT at 55C from less than 5 min to 30 min in the presence of template-primer. In addition, these mutations alter the thermal profile by increasing specific activity of the pentuple mutant (M5) over a broad range of cDNA synthesis temperatures (2570C). We further show that M5 generates higher cDNA yields and exhibits better RTPCR performance compared to wild-type RT when used at high temperature to amplify RNA targets containing secondary structure. Finally, we demonstrate that M5 exhibits tighter binding (lower K-m) to template-primer, which likely protects against heat inactivation.
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