Wheeler Amanda, Czado Natalia, Gangitano David, Turnbough Meredith, Hughes-Stamm Sheree
Department of Forensic Science, College of Criminal Justice, Sam Houston State University, Huntsville, TX, USA.
QIAGEN Inc., 19300 Germantown Road, Germantown, MD, 20874, USA.
Int J Legal Med. 2017 Jan;131(1):61-66. doi: 10.1007/s00414-016-1405-5. Epub 2016 Jun 23.
Formalin fixation is commonly used to preserve tissue sections for pathological testing and embalming cadavers for medical dissection or burial. DNA extracted from formalin-fixed tissues may also provide an alternative source of genetic material for medical diagnosis and forensic casework, such as identifying unknown embalmed human remains. Formaldehyde causes DNA damage, chemical modifications, and degradation, thereby reducing the quantity and quality of DNA available for downstream genetic analyses. By comparing the DNA yield, level of DNA degradation, and short tandem repeat (STR) success of various tissue types, this study is the first of its kind to provide some guidance on which samples from embalmed bodies are likely to generate more complete STR profiles. Tissue samples were dissected from three male embalmed cadavers and included bone, cartilage, hair, muscle, internal organs, skin, teeth, and nail clippings. DNA was purified from all samples using the QIAamp® FFPE Tissue Kit (Qiagen), quantified using the QuantiFiler® Trio DNA Quantification kit (Life Technologies), and genotyped using the GlobalFiler® PCR Amplification Kit (Life Technologies). Results of this study showed variation in DNA quantity and STR success between different types of tissues and some variation between cadavers. Overall, bone marrow samples resulted in the highest DNA yields, the least DNA degradation, and greatest STR success. However, several muscle, hair, and nail samples generated higher STR success rates than traditionally harvested bone and tooth samples. A key advantage to preferentially using these tissue samples over bone (and marrow) and teeth is their comparative ease and speed of collection from the cadaver and processing during DNA extraction. Results also indicate that soft tissues affected by lividity (blood pooling) may experience greater exposure to formalin, resulting in more DNA damage and reduced downstream STR success than tissues under compression. Overall, we recommend harvesting from selected muscles (gastrocnemius, rectus femoris, flexor digitorum brevis, masseter, brachioradialis) or fingernails for human identification purposes.
福尔马林固定常用于保存组织切片以进行病理检测,以及对尸体进行防腐处理以用于医学解剖或埋葬。从福尔马林固定的组织中提取的DNA也可为医学诊断和法医案件工作提供另一种遗传物质来源,例如识别身份不明的防腐人类遗骸。甲醛会导致DNA损伤、化学修饰和降解,从而减少可用于下游遗传分析的DNA数量和质量。通过比较各种组织类型的DNA产量、DNA降解水平和短串联重复序列(STR)成功率,本研究首次为来自防腐尸体的哪些样本可能产生更完整的STR图谱提供了一些指导。从三具男性防腐尸体上解剖组织样本,包括骨骼、软骨、毛发、肌肉、内脏器官、皮肤、牙齿和指甲剪。使用QIAamp® FFPE组织试剂盒(Qiagen)从所有样本中纯化DNA,使用QuantiFiler® Trio DNA定量试剂盒(Life Technologies)进行定量,并使用GlobalFiler® PCR扩增试剂盒(Life Technologies)进行基因分型。本研究结果表明,不同类型组织之间的DNA数量和STR成功率存在差异,尸体之间也存在一些差异。总体而言,骨髓样本的DNA产量最高,DNA降解最少,STR成功率最高。然而,一些肌肉、毛发和指甲样本产生的STR成功率高于传统采集的骨骼和牙齿样本。优先使用这些组织样本而非骨骼(和骨髓)及牙齿进行检测的一个关键优势在于,从尸体采集这些样本以及在DNA提取过程中进行处理相对更容易、速度更快。结果还表明,受尸斑(血液淤积)影响的软组织可能比受压组织接触到更多福尔马林,从而导致更多DNA损伤和下游STR成功率降低。总体而言,我们建议为了身份鉴定目的,从选定的肌肉(腓肠肌、股直肌、趾短屈肌、咬肌、肱桡肌)或指甲上采集样本。