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Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer.

Authors
  • 1
Type
Published Article
Journal
Nucleic acids research
1362-4962
Publication Date
Volume
37
Issue
2
Pages
473–481
Identifiers
DOI: 10.1093/nar/gkn952
PMID: 19056821
Source
Medline

Abstract

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 55 degrees C from less than 5 min to approximately 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 (25-70 degrees C). We further show that M5 generates higher cDNA yields and exhibits better RT-PCR 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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