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Establishment of novel identification systems for biofluids by DNA methylation profiling
|關鍵字:||體液;混和體液;DNA甲基化;甲基化特異性PCR(MSP)結合單一鹼基延長(SBE);甲基化敏感限制內切酶PCR(MSRE-PCR);miniSTR型別;biofluids;mixtures;DNA methylation;MSP-SBE;MSRE-PCR;miniSTR||引用:|| K. Virkler, I.K. Lednev, Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene, Forensic Sci Int 188 (2009) 1-17.  S.S. Tobe, N. Watson, N.N. Daeid, Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA, J Forensic Sci 52 (2007) 102-109.  M. Cox, A study of the sensitivity and specificity of four presumptive tests for blood, J Forensic Sci 36 (1991) 1503-1511.  J.P. de Almeida, N. Glesse, C. Bonorino, Effect of presumptive tests reagents on human blood confirmatory tests and DNA analysis using real time polymerase chain reaction, Forensic Sci Int 206 (2011) 58-61.  G.M. Willott, An improved test for the detection of salivary amylase in stains, J Forensic Sci Soc 14 (1974) 341-344.  P.H. 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鑑定現場體液在刑事鑑定中扮演非常重要的角色，而體液型別可藉由分析特定基因標記的甲基化狀態來鑑定為何種體液。本研究搜尋Infinium HumanMethylation450 BeadChip資料庫中，找出對精液、陰道液、唾液、周邊血及月經血具甲基化特異性的基因標記，並發展兩套可用來分析這些基因標記甲基化狀態的分析系統。
第二套分析系統是利用甲基化敏感限制內切酶PCR(MSRE-PCR) 技術，此系統為可同時分析10個基因標記的多重引子MSRE-PCR，共包含8個體液特異性甲基化基因標記及2個控制基因標記。在分析5種不同的體液樣本測試中，均可無誤的鑑定出其體液型別。另外進行本系統之靈敏性、再現性及混合樣品等之確效性實驗，也都呈現良好且正確的結果。此外，此系統可和AmpFlSTR® MiniFiler™ PCR Amplification Kit在同一反應中一起進行，同時產出體液鑑定基因標記之甲基化狀態和miniSTR型別，達成同時鑑定體液型別和鑑定身分之效果。
The identification of a specific biofluid encountered in a forensic investigation can give crucial information. This identification can be aided by methylation profiles based on selected markers specific to a range of biofluids. In this study, the open database of Infinium HumanMethylation450 BeadChip was searched for markers specific for semen, vaginal fluids, saliva, venous blood and menstrual blood. Two systems based on analyzing methylation profile were developed.
The first system utilizes methylation-specific PCR combined with single-base-extension (MSP-SBE). A total of eight biofluid-specific methylated markers for saliva, venous blood, vaginal fluids and semen were isolated. These biofluid-specific markers, a control marker to confirm bisulfite conversion, and a gender marker were incorporated into a 10-plex methylation-specific PCR single-base-extension (MSP-SBE) system. Analysis of DNA samples isolated from various biofluids that had been treated with bisulfite, resulted in all eight markers detecting the biofluid to which they were designed. Unambiguous biofluid identification occurred from both single sources of biofluids and complex mixtures. The efficacy of the assay and application to forensic practice was demonstrated using five non-probative samples from real alleged sexual assault cases. The system unambiguously determined the biofluid types for the non-probative forensic samples that previously resulted in inconclusive or conflicting results using traditional tests.
The second system employs methylation sensitive restriction enzyme-PCR (MSRE-PCR). A total of 8 biofluid-specific methylated markers and 2 control markers were combined into a 10-plex methylation sensitive restriction enzyme-PCR (MSRE-PCR) system. Based upon the analysis of DNA samples from 5 various biofluid types, unambiguous results were obtained to identify the biofluid from which it originated. Validation studies of the developed 10-plex MSRE-PCR included sensitivity, reproducibility and mixed biofluids. Co-amplification of the established MSRE-PCR system and the microsatellite loci in AmpFlSTR® MiniFiler™ PCR Amplification Kit was performed to generate both the methylation profile for biofluid type and the miniSTR profile. This allowed human identification and the identification of the biofluid type to be performed in a single reaction.
This study established two systems analyzing methylation profile for identification of biofluids. Both systems displayed accuracy, sensitivity and feasibility for forensic application.
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