Extreme PCR: Efficient and Specific DNA Amplification in 15-60 Seconds
Extreme PCR: Efficient and Specific DNA Amplification in 15-60 Seconds
复制标题
DOI：
10.1373/clinchem.2014.228304
复制
发表时间：
2015-01-01
影响因子：
9.3
通讯作者：
Wittwer, Carl T.
中科院分区：
文献类型：
--
作者：
Farrar, Jared S.;Wittwer, Carl T.
背景技术背景：PCR是分子生物学和诊断学中的关键技术，通常在大约一小时内扩增和定量特定的DNA片段。然而，PCR的动力学极限是unknown.METHODS：我们开发了原型仪器，在退火/延伸温度为62摄氏度-76摄氏度和变性温度为85摄氏度-92摄氏度的0.4-2.0秒的温度循环1- 5-μ L的样品。引物和聚合酶浓度增加到典型浓度的10至20倍，以使引物退火和聚合酶延伸的动力学与更快的温度循环相匹配。我们通过琼脂糖凝胶和高分辨率熔解分析评估了分析特异性和产率。通过实时光学监测证实了扩增效率和分析灵敏度。结果：使用来自人类基因组DNA的单拷贝基因，我们在15-60 s内扩增了45- 102-bp的靶标。琼脂糖凝胶显示明亮的单一条带在预期的大小，和高分辨率的熔解曲线显示单一的产品，而不使用任何“热启动”技术。采用0.8 ~ 1.9s的循环，扩增效率为91.7%~ 95.8%。一个60-bp的基因组目标的扩增在14.7秒，通过使用35个cycles.CONCLUSIONS：PCR所需的时间是负相关的关键反应物的浓度。通过将引物和聚合酶浓度增加10- 20倍，温度循环为0.4-2.0 s，可以从人DNA中进行有效（>90%）、特异性、高产率的PCR，
BACKGROUND: 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.METHODS: We developed prototype instruments to temperature cycle 1- to 5-mu L samples in 0.4-2.0 s at annealing/extension temperatures of 62 degrees C-76 degrees C and denaturation temperatures of 85 degrees C-92 degrees 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.RESULTS: 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.CONCLUSIONS: 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