Plastic versus glass capillaries for rapid-cycle PCR
Plastic versus glass capillaries for rapid-cycle PCR
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DOI：
10.2144/000112722
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发表时间：
2008-04-01
期刊：
影响因子：
2.7
通讯作者：
Wittwer, Carl T.
中科院分区：
文献类型：
--
作者：
Elenitoba-Johnson, Oluwole;David, Derek;Wittwer, Carl T.
快速循环PCR采用快速的温度转变和最小的变性和退火时间为“0”秒，在10至30分钟内完成30个循环。最受欢迎的平台放大样品的光泽毛细血管周围安排的旋转木马与循环空气温度控制。最近，塑料毛细管代替玻璃毛细管成为可能。我们比较了塑料和玻璃毛细管用于快速循环PCR的性能。与玻璃毛细管相比，更厚的壁、更低的导热率和更低的表面积体积比减慢了塑料毛细管的传热速度。玻璃毛细管中的样品达到了变性和退火目标温度，而塑料毛细管中的样品低于目标温度6 - 7℃。对两个人类基因组靶点（APOE和ACVRL1）和一个质粒（pBR322）进行快速循环PCR，扩增长度为225 - 300 bp的片段，熔化温度为90.3℃- 93.1℃。终点凝胶分析显示，样品在玻璃中有很强的特异性扩增，而在塑料中完全没有扩增。通过延长变性和退火时间至5或10 s，只有APOE目标被成功扩增。为了达到塑料毛细血管的目标温度，需要20秒的保温时间。
Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of "0" s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in gloss capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries become available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area - to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees - 7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225 - 300 bp in length with melting temperatures of 90.3 degrees - 93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specfic amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach tat-get temperatures it plastic capillaries.