背景:数字聚合酶链反应(数字PCR)是生命科学研究和临床诊断中重要的定量核酸分析方法。一个重要的假设是通过将单个核酸分子物理上限制在小体积中,可以增加相对浓度,从而进一步提高分析性能,这通常被定义为数字PCR中的限制效应。然而,这种限制效应的实验研究可能是具有挑战性的,因为它需要可以产生不同体积的分区的微流体装置和可以监测扩增动力学的仪器。(96).
结果:这里,我们开发了一种实时数字PCR系统,该系统具有多卷液滴阵列滑动芯片(Muda-SlipChip),可以产生125nL的液滴,25nL,5nL,和1nL通过简单的“负载滑移”操作。在数字区域,通过减少音量,相对浓度增加,可以加速放大动力学,以及达到荧光阈值的时间,或Cq值,可以减少。当每个孔的拷贝数远高于1时,相对浓度与分区体积无关,因此,扩增动力学在不同体积分区中是相似的。该系统不仅限于研究数字核酸扩增的动力学,它还可以通过组合数字和模拟定量算法将数字核酸分析的动态范围扩展另外三个数量级。(140).
结论:在这项研究中,我们首次通过具有多卷液滴阵列滑动芯片(Muda-SlipChip)的新型实时数字PCR系统,通过实验研究了数字PCR社区中的限制效应。并且通过该系统验证了与常规数字PCR相比更宽的定量方法的动态范围。该系统为生命科学研究和临床诊断提供了新的机会。(63).
BACKGROUND: Digital polymerase chain reaction (digital PCR) is an important quantitative nucleic acid analysis method in both life science research and clinical diagnostics. One important hypothesis is that by physically constraining a single nucleic acid molecule in a small volume, the relative concentration can be increased therefore further improving the analysis performance, and this is commonly defined as the
confinement effect in digital PCR. However, experimental investigation of this
confinement effect can be challenging since it requires a microfluidic device that can generate partitions of different volumes and an instrument that can monitor the kinetics of amplification. (96).
RESULTS: Here, we developed a real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip) that can generate droplet of 125 nL, 25 nL, 5 nL, and 1 nL by a simple \"load-slip\" operation. In the digital region, by reducing the volume, the relative concentration is increased, the amplification kinetic can be accelerated, and the time to reach the fluorescence threshold, or Cq value, can be reduced. When the copy number per well is much higher than one, the relative concentration is independent of the partition volume, thus the amplification kinetics are similar in different volume partitions. This system is not limited to studying the kinetics of digital nucleic acid amplification, it can also extend the dynamic range of the digital nucleic acid analysis by additional three orders of magnitude by combining a digital and an analog quantification algorithm. (140).
CONCLUSIONS: In this study, we experimentally investigated for the first time the
confinement effect in the community of digital PCR via a new real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip). And a wider dynamic range of quantification methods compared to conventional digital PCR was validated by this system. This system provides emerging opportunities for life science research and clinical diagnostics. (63).