Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; Current affiliation: MD-PhD Program, Virginia Commonwealth University, Richmond, VA.
Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT;
Clin Chem. 2015 Jan;61(1):145-53. doi: 10.1373/clinchem.2014.228304. Epub 2014 Oct 15.
PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown.
We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4-2.0 s at annealing/extension temperatures of 62 °C-76 °C and denaturation temperatures of 85 °C-92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring.
Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15-60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any "hot start" technique. Amplification efficiencies were 91.7%-95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles.
The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4-2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.
PCR 是分子生物学和诊断学的关键技术，通常在大约 1 小时内扩增和定量特定的 DNA 片段。然而，PCR 的动力学限制尚不清楚。
我们开发了原型仪器，可在 0.4-2.0 秒内对 1-5μL 样品进行温度循环，退火/延伸温度为 62°C-76°C，变性温度为 85°C-92°C。引物和聚合酶的浓度增加了 10-20 倍，超过了典型浓度，以匹配引物退火和聚合酶延伸的动力学与更快的温度循环。我们通过琼脂糖凝胶电泳和高分辨率熔解分析评估分析特异性和产量。通过实时光学监测证明了扩增效率和分析灵敏度。
使用来自人类基因组 DNA 的单拷贝基因，我们在 15-60 秒内扩增了 45-102bp 的目标。琼脂糖凝胶显示出预期大小的明亮单带，高分辨率熔解曲线显示出无需使用任何“热启动”技术的单一产物。使用 0.8-1.9 秒的循环和单分子灵敏度，扩增效率为 91.7%-95.8%。通过使用 35 个循环，在 14.7 秒内扩增了 60bp 的基因组靶标。
PCR 所需的时间与关键反应物的浓度成反比。通过将引物和聚合酶的浓度提高 10-20 倍，同时使用 0.4-2.0 秒的温度循环，可以在<15 秒内从人类 DNA 中进行高效（>90%）、特异性、高产量的 PCR。极限 PCR 证明了即时检测传染病、法医学和任何可能关键的即时结果的应用的可行性。
