微流控数字 PCR 与常规定量 PCR 测量拷贝数变异的比较。

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.

机构信息

LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.

出版信息

Nucleic Acids Res. 2012 Jun;40(11):e82. doi: 10.1093/nar/gks203. Epub 2012 Feb 28.

Abstract

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

摘要

数字 PCR(dPCR)的一个优势是具有无与伦比的精度潜力,能够实现更小的倍数变化测量。可以从这种改进的精度中受益的一个评估示例是测量患者血浆中无细胞 DNA(cfDNA)部分的肿瘤相关拷贝数变异(CNV)。为了研究 dPCR 的潜在精度,并将其与已建立的定量 PCR(qPCR)技术进行比较,我们使用乳腺癌细胞系来研究 HER2 基因扩增,并对一系列不同的 CNV 进行建模。我们表明,在同等实验重复的情况下,dPCR 可以测量到比 qPCR 更小的 CNV。由于 dPCR 精度直接取决于重复测量的数量和模板浓度,因此我们还开发了一种方法来协助设计用于测量 CNV 的 dPCR 实验。我们使用现有的模型(基于泊松和二项式分布)来推导出 dPCR 中固有的方差表达式,然后进行了功率计算,以确定在给定模板浓度下可靠检测给定倍数变化所需的实验规模。这项工作将促进 dPCR 在癌症诊断和 cfDNA 分析等关键诊断应用中的未来转化。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/bfe8/3367212/39b57858f70e/gks203f1.jpg

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