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Purification of crime scene DNA extracts using centrifugal filter devices

机译：使用离心过滤装置纯化犯罪现场的DNA提取物

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Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values P-values Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used.
机译：背景技术法医DNA分析的成功受到犯罪现场发现的生物学证据的大小，质量和纯度的限制。样品中的杂质会抑制PCR，导致部分或负面的DNA分布。各种DNA纯化方法被应用于去除杂质，例如，采用离心过滤装置。但是，不管使用哪种方法，DNA纯化都会导致DNA丢失。在这里，我们针对各种抑制PCR的犯罪现场样本的回收率和一般性能评估了Amicon Ultra 30 K和Microsep 30 K过滤器设备。方法采用定量PCR收集使用Amicon Ultra 30 K和Microsep 30 K纯化DNA的回收率。使用定量PCR和短串联重复序列（STR）分析法分析了模拟犯罪现场的DNA提取物，以测试这两种过滤器设备的总体性能和去除抑制剂的性能。此外，评估了使用每种设备进行的长期常规病例DNA分析的结果。结果使用Microsep 30 K，可回收14至32％的输入DNA，而Amicon Ultra 30 K可保留62至70％的DNA。过滤器纯化后纯度的提高抵消了部分DNA损失，从而导致牛仔布上的血液（Amicon Ultra 30 K和Microsep 30 K）和包膜上的唾液（Amicon Ultra 30 K）的电泳峰高度略有增加。比较Amicon Ultra 30 K和Microsep 30 K用于纯化模拟犯罪现场样品的DNA提取物，前者在强奸案样品中产生明显更高的峰高（P值P值结论结论：Amicon Ultra 30 K的性能优于Microsep 30 K，原因是以获得更高的DNA回收率和更有效地去除PCR抑制性物质。过滤器设备的不同性能可能是由过滤器和塑料制品的质量（例如，它们的DNA结合特性）引起的。对不纯犯罪现场样本进行成功的DNA分析以及在基于PCR的不同分析系统之间进行一致性（例如定量和STR分析）所必需的，为了最大程度地获得完整的STR DNA图谱并创建有效的工作流程，进行的DNA纯化应与所用STR分析系统的抑制剂耐受性相关。

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