期刊
BIOCHEMISTRY
卷 57, 期 31, 页码 4607-4619出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.8b00200
关键词
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资金
- Air Force Office of Scientific Research [FA9550-14-1-0089]
- National Science Foundation [1541244]
- National Institutes of Health [1T32LM012414-01A1]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1541244] Funding Source: National Science Foundation
Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 degrees C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.
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