在法医犯罪现场调查中,血液等生物流体常见于土壤中。然而,由于抑制剂的存在,限制了产生可报告的DNA图谱所需的脱氧核糖核酸(DNA)的有效提取和扩增,因此对血染土壤的分析可能具有挑战性。法医学中有一些提取方法已应用于血染土壤,但是这些产生了零星的结果。这项研究从古代DNA和环境DNA领域采取了许多不同的提取方法,并将它们分解为预处理的各个步骤,孵化,分离和纯化。独立评估这些步骤,然后将其结合到各种提取方法中,以确定可以有效且可靠地从血液染色的土壤中分析人DNA的最佳技术。测试涉及评估三种提取缓冲液,(十六烷基三甲基溴化铵,硫氰酸胍,和蛋白酶K),四种预处理方法,(聚乙烯吡咯烷酮,乙二胺四乙酸,盐酸,和氢氧化钠),三个分离步骤,(离心,苯酚氯仿,和氯仿)和四个纯化步骤,(尺寸排阻色谱法,绑定洗脱柱,异丙醇沉淀和二氧化硅磁珠)。发现最有效的程序是用蛋白酶K提取缓冲液进行聚乙烯吡咯烷酮预处理,然后在离心或不离心的情况下进行磁性二氧化硅珠纯化。然而,发现离心分离在预处理步骤后与在孵育步骤后同样有效。我们的结果表明,大多数当前的法医程序将受益于在通过自动化DNA谱分析流程进行处理之前添加的预处理步骤。
In forensic crime scene investigations, biological fluids such as blood are commonly found in soil. However, the analysis of blood-stained soil can be challenging due to the presence of inhibitors which limit the effective extraction and amplification of the deoxyribonucleic acid (DNA) required to produce a reportable DNA profile. There are some extraction methods that have been applied to blood-stained soil in forensic science, but these have produced sporadic results. This research has taken a number of different extraction methods from the fields of ancient DNA and environmental DNA and broken them down into the individual steps of pre-treatment, incubation, separation and purification. These steps were assessed independently then combined into various extraction methods to determine the best technique that can effectively and reliably profile human DNA from blood-stained soil. Testing involved assessment of three extraction buffers, (cetyltrimethylammonium bromide, guanidine thiocyanate, and proteinase K), four pre-treatment methods, (polyvinylpyrrolidone, ethylenediaminetetraacetic acid, hydrochloric acid, and sodium hydroxide), three separation steps, (centrifugation, phenol chloroform, and chloroform) and four purification steps, (size exclusion chromatography, bind elute columns, isopropanol precipitation and silica magnetic beads). The most effective procedure was found to be a polyvinylpyrrolidone pre-treatment with a proteinase K extraction buffer followed by magnetic silica bead purification with or without centrifugation. However, centrifugation separation was found to be equally effective after the pre-treatment step as after the incubation step. Our results shows that most of the current forensic procedures would benefit from the addition of a pre-treatment step prior to processing through the automated DNA profiling pipeline.