Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA
Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA
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DOI：
10.1021/ac980452i
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发表时间：
1998-10-15
影响因子：
7.4
通讯作者：
Landers, JP
中科院分区：
文献类型：
--
作者：
Oda, RP;Strausbauch, MA;Landers, JP
人们对通过毛细管电泳提高 DNA 分析速度的兴趣促使人们努力将 DNA 扩增集成到微型设备中。这是很难实现的，因为有效的聚合酶链式反应 (PCR) 所需的热循环取决于加热源和 PCR 混合物容器之间的有效接触。我们描述了一种在电泳芯片状玻璃室中快速有效地热循环 PCR 混合物的非接触方法。热循环是通过使用钨灯作为廉价的红外辐射源来介导的，并通过电磁门压缩空气源进行冷却。在玻璃微腔中以 10 摄氏度/秒（加热）和 20 摄氏度/秒（冷却）的速度在 94 至 55 摄氏度之间升温，可以实现快至 17 秒的循环时间。使用 T 细胞受体 β 链特异性引物成功进行了基因组 DNA 扩增，并且可以在商业 PCR 仪器所需时间的一小部分内生成可检测的产物。未发现非接触介导的热循环形式仅限于单个 DNA 片段扩增。将热循环方法成功应用于定量竞争性 PCR（同时扩增靶标 DNA 和竞争者 DNA）和循环测序反应（同时扩增双脱氧终止片段）。这为将 DNA 热循环实施到各种微加工形式中以实现快速 PCR 片段识别和 DNA 测序奠定了基础。
Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.