Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.
Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.
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DOI：
10.1038/srep13174
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发表时间：
2015-08-25
影响因子：
4.6
通讯作者：
Fu B
中科院分区：
文献类型：
--
作者：
Dong L;Meng Y;Sui Z;Wang J;Wu L;Fu B
数字聚合酶链式反应(DPCR)是一种不使用外部标准品来测量目标DNA绝对拷贝数的独特方法。然而，必须解决不同dPCR平台在DNA拷贝数测量方面的可比性，然后才能从根本上将dPCR归类为绝对定量技术。4个dPCR平台的准确性和测量不确定度方面的可比性通过使用认证的质粒标准物质进行了研究。研究发现，与基于芯片的QuantStudio12k或BioMark不同，质粒构象对基于液滴的dPCR(QX100和RAINDOP)有显著影响。BioMark、QX100、QuantStudio12k和RAUNDROP的分配体积的相对不确定度分别为0.7%、0.8%、2.3%和2.9%。使用四个dPCR平台对认证的pNIM-001质粒进行的测量对分配体积进行了校正，并在扩展的不确定度内与认证值非常一致。这表明这四个dPCR平台在定量DNA拷贝数方面具有相当的有效性。这些发现为在使用不同的dPCR平台时确定DNA拷贝数的方法提供了独立的评估，并强调了在设计dPCR实验时应考虑的重要因素。
Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.