Gleerup David, Chen Yao, Van Snippenberg Willem, Valcke Cedric, Thas Olivier, Trypsteen Wim, De Spiegelaere Ward
Laboratory of Veterinary Morphology, Ghent University, Belgium; Ghent University Digital PCR Consortium, Ghent University, Belgium.
Ghent University Digital PCR Consortium, Ghent University, Belgium; Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Belgium.
Anal Chim Acta. 2023 Oct 23;1279:341822. doi: 10.1016/j.aca.2023.341822. Epub 2023 Sep 15.
Accurate methods to assess DNA integrity are needed for many biomolecular methods. A multiplex digital PCR (dPCR) method designed for interspaced target sequences can be used to assess sequence integrity of large DNA strands. The ratio of single positive partitions versus double positive partitions is then used to calculate the sheared DNA strands. However, this simple calculation is only valid with low DNA concentration. We here describe a method based on probability calculations which enables DNA quality analysis in a large dynamic range of DNA concentrations.
Known DNA integrity percentages were mimicked using artificial double stranded DNA in low, intermediate and high DNA concentration scenarios, respectively 600, 12500 and 30000 copies of DNA per reaction. At low concentrations both methods were similar. However, at the intermediate concentration (12500 copies per reaction) the ratio based method started producing a larger error than the proposed probability calculation method with a mean relative error of 20.7 and 16.7 for the Bruner and the proposed method respectively. At the high concentration (30000 copies per reaction) only the proposed method provided accurate measurements with a mean relative error of 60.9 and 9.3 for the ratio based and the proposed method respectively. Furthermore, while both methods have a bias, it is constant for the proposed method, while it decreases with the integrity of the DNA for the ratio based method. The probability calculation equation was extended to 4 dimensions and a proof of concept experiment was performed, the data suggested that the 4 dimensional equation is valid.
We here validate a method of estimating DNA integrity with dPCR using multiple probe combinations, allowing fast and flexible DNA integrity analysis. Additionally, we extend the method from 2 to 4 plex for more accurate DNA integrity measurements.
许多生物分子方法都需要准确评估DNA完整性的方法。一种针对间隔靶序列设计的多重数字PCR（dPCR）方法可用于评估大DNA链的序列完整性。然后，使用单阳性分区与双阳性分区的比率来计算剪切的DNA链。然而，这种简单的计算仅在低DNA浓度下有效。我们在此描述一种基于概率计算的方法，该方法能够在大动态范围的DNA浓度中进行DNA质量分析。
分别在低、中、高DNA浓度情况下，即每个反应600、12500和30000个DNA拷贝，使用人工双链DNA模拟已知的DNA完整性百分比。在低浓度下，两种方法相似。然而，在中等浓度（每个反应12500个拷贝）时，基于比率的方法开始产生比所提出的概率计算方法更大的误差，布鲁纳方法和所提出方法的平均相对误差分别为20.7和16.7。在高浓度（每个反应30000个拷贝）时，只有所提出的方法能提供准确的测量结果，基于比率的方法和所提出方法的平均相对误差分别为60.9和9.3。此外，虽然两种方法都存在偏差，但所提出方法的偏差是恒定的，而基于比率的方法的偏差会随着DNA的完整性而降低。概率计算方程扩展到了4维，并进行了概念验证实验，数据表明4维方程是有效的。
我们在此验证了一种使用多种探针组合通过dPCR估计DNA完整性的方法，可实现快速灵活的DNA完整性分析。此外，我们将该方法从2重扩展到4重，以进行更准确的DNA完整性测量。
