Forensic Genetics Research Group, Oslo University Hospital, Oslo, Norway; Department of Forensic Medicine, University of Oslo, Oslo, Norway.
Forensic Genetics Research Group, Oslo University Hospital, Oslo, Norway.
Forensic Sci Int Genet. 2022 Nov;61:102777. doi: 10.1016/j.fsigen.2022.102777. Epub 2022 Sep 27.
The application of qPCR to estimate the quantity of DNA present is usually based upon a short amplicon (typically c.80bp) and a longer amplicon (typically c.200-300bp) where the latter is used to determine the amount of degradation present in a sample. The data are used to make decisions about a) whether there is sufficient template to amplify? b) how much of the elution volume to forward to PCR? A typical multiplex amplifies template in the region of 100-500bp. Consequently, the results from an 80bp amplicon will tend to overestimate the actual amplifiable quantity that is present in a degraded sample. To compensate, a method is presented that relates the quantity of amplifiable DNA to the average RFU of the amplified fragments. This provides greatly improved accuracy of the estimated quantity of DNA present, which may differ by more than an order of magnitude compared to qPCR. The relative DNA quantities can be apportioned per contributor once mixture proportions are ascertained with probabilistic genotyping software (EuroForMix). The motivation for this work was to provide an improved method to generate data to prepare distributions that are used to inform activity level propositions. However, other applications will benefit, particularly those where extraction and quantification are bypassed: For example direct PCR and Rapid DNA technology. The overall aim of this work was to provide a method of quantification that is standardised and can be used to compare results between different laboratories that use different multiplexes. A software solution "ShinyRFU" is provided to aid calculations.
qPCR 通常用于估计 DNA 数量，其应用基于短扩增子（通常为 80bp）和长扩增子（通常为 200-300bp），后者用于确定样品中降解的程度。数据用于做出以下决定：a）是否有足够的模板进行扩增？b）要向 PCR 传递多少洗脱体积？典型的多重扩增模板位于 100-500bp 范围内。因此，80bp 扩增子的结果往往会高估降解样品中实际可扩增的数量。为了弥补这一点，提出了一种将可扩增 DNA 数量与扩增片段的平均 RFU 相关联的方法。这大大提高了估计存在的 DNA 数量的准确性，与 qPCR 相比，其差异可能超过一个数量级。一旦用概率基因分型软件（EuroForMix）确定了混合物的比例，就可以按每个贡献者分配相对 DNA 数量。这项工作的动机是提供一种改进的方法来生成数据，以准备用于告知活动水平提案的分布。然而，其他应用也将受益，特别是那些绕过提取和定量的应用：例如直接 PCR 和 Rapid DNA 技术。这项工作的总体目标是提供一种标准化的定量方法，可以用于比较使用不同多重扩增的不同实验室之间的结果。提供了一个名为“ShinyRFU”的软件解决方案来辅助计算。
