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Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication
Biochemistry ( IF 2.9 ) Pub Date : 2018-04-09 00:00:00 , DOI: 10.1021/acs.biochem.8b00200 John N. Milligan 1 , Raghav Shroff 1 , Daniel J. Garry 1 , Andrew D. Ellington 1
Biochemistry ( IF 2.9 ) Pub Date : 2018-04-09 00:00:00 , DOI: 10.1021/acs.biochem.8b00200 John N. Milligan 1 , Raghav Shroff 1 , Daniel J. Garry 1 , Andrew D. Ellington 1
Affiliation
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 °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.
中文翻译:
高温等温区室自复制的嗜热链置换聚合酶的进化。
置换链的聚合酶是等温扩增(IA)反应的关键组成部分,其中缺乏热循环可减少设备需求并缩短响应时间,特别是对于即时医疗应用而言。为了改善链置换聚合酶的功能,我们已经开发了一种基于乳液的定向进化方案,即高温等温区室自我复制(HTI-CSR),它不依赖于热循环。从算法优化的改组后的库中提取出嗜热脂肪地芽孢杆菌(Bst LF)和水生栖热菌的核酸外切酶缺陷型Family A聚合酶(Klentaq),我们已经应用HTI-CSR产生了更热稳定的置换链的聚合酶变体,即使在高达95°C的热挑战导致更好的引物退火后,其在环介导的等温扩增和滚环扩增中也表现良好。新酶(v5.9)还能够进行多种新反应,包括等温超支化滚环扩增。现在应证明HTI-CSR方法可用于发展链置换聚合酶中其他有益的表型。
更新日期:2018-04-09
中文翻译:
高温等温区室自复制的嗜热链置换聚合酶的进化。
置换链的聚合酶是等温扩增(IA)反应的关键组成部分,其中缺乏热循环可减少设备需求并缩短响应时间,特别是对于即时医疗应用而言。为了改善链置换聚合酶的功能,我们已经开发了一种基于乳液的定向进化方案,即高温等温区室自我复制(HTI-CSR),它不依赖于热循环。从算法优化的改组后的库中提取出嗜热脂肪地芽孢杆菌(Bst LF)和水生栖热菌的核酸外切酶缺陷型Family A聚合酶(Klentaq),我们已经应用HTI-CSR产生了更热稳定的置换链的聚合酶变体,即使在高达95°C的热挑战导致更好的引物退火后,其在环介导的等温扩增和滚环扩增中也表现良好。新酶(v5.9)还能够进行多种新反应,包括等温超支化滚环扩增。现在应证明HTI-CSR方法可用于发展链置换聚合酶中其他有益的表型。