Quantitative polymerase chain reaction using infrared heating on a microfluidic chip.
Quantitative polymerase chain reaction using infrared heating on a microfluidic chip.
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DOI：
10.1021/ac203307h
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发表时间：
2012-03-20
影响因子：
7.4
通讯作者：
Roper, Michael G.
中科院分区：
文献类型：
--
作者：
Yu, Yingjie;Li, Bowei;Baker, Christopher A.;Zhang, Xinyu;Roper, Michael G.
微型装置中的IR介导的PCR是用于快速扩增核酸的已建立的技术。在这份报告中，我们已经扩大了适用性的IR-PCR的起始拷贝数的定量测定，在扩增过程中整合荧光检测。将微流体装置放置在IR长通滤波器和热镜之间将背景降低到使得能够在整个热循环过程中进行荧光测量的水平。延伸步骤期间的平均荧光强度显示出指数增加的预期趋势，随后是连续循环中的平台期。扩增不同起始拷贝数的PUC 19模板，并且阈值循环显示起始DNA的量减少而增加。扩增效率为80%，凝胶分离表明没有检测到非特异性产物。使用IR加热产生解链曲线，并指示304 bp扩增子的解链温度为85 °C，其与使用常规PCR系统获得的解链温度相当。该方法将适用于其他类型的IR介导的扩增系统，例如等温扩增和结合了PCR前和PCR后过程的联合收割机的高度集成系统。
IR-mediated PCR in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR longpass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 template at different starting copy numbers were amplified and the threshold cycle showed an increase for decreasing amounts of starting DNA. Amplification efficiency was 80% and gel separation indicated no detectable non-specific product. A melting curve was generated using IR heating and indicated a melting temperature of 85 °C for the 304 bp amplicon which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification and in highly integrated systems that combine pre- and post-PCR processes.
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影响因子：
7.4
作者：
Zhang, Xinyu;Roper, Michael G.
通讯作者：
Roper, Michael G.
影响因子：
7.4
作者：
Roper, Michael G.;Easley, Christopher J.;Landers, James P.
通讯作者：
Landers, James P.
影响因子：
2.8
作者：
Walsh, EJ;King, C;Gonzalez, A
通讯作者：
Gonzalez, A
影响因子：
7.4
作者：
Lagally, ET;Medintz, I;Mathies, RA
通讯作者：
Mathies, RA
DOI：
10.1073/pnas.93.11.5556
发表时间：
1996-05-28
影响因子：
11.1
作者：
Burns, MA;Mastrangelo, CH;Burke, DT
通讯作者：
Burke, DT