Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system
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ORIGINAL PAPER
Novel identification of biofluids using a multiplex methylationspecific PCR combined with single-base extension system Yu-Chih Lin1,2 • Li-Chin Tsai3 • James Chun-I Lee4 • Kuo-Lan Liu3,5 Jason Tze-Cheng Tzen1 • Adrian Linacre6 • Hsing-Mei Hsieh3
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Accepted: 2 March 2016 Ó Springer Science+Business Media New York 2016
Abstract Purpose Knowledge of the composition of complex body fluid mixtures may aid forensic investigations greatly. However, many of the traditional tests are presumptive in nature and can lead to ambiguous results. The aim of this study is to establish a reliable method to identify various biofluids via analysis of their DNA methylation profiles. Methods A total of eight biofluid-specific methylated markers for saliva, venous blood, vaginal fluids, and semen were isolated from the open database of Infinium HumanMethylation450 BeadChip. These biofluid-specific markers, a control marker to confirm bisulfite conversion,
Electronic supplementary material The online version of this article (doi:10.1007/s12024-016-9763-3) contains supplementary material, which is available to authorized users. & Hsing-Mei Hsieh [email protected] 1
Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo Kuang Road, South District, Taichung City 40227, Taiwan, ROC
2
Taichung City Government Police Department, 588 Wenxin Road Section 2, Xitun District, Taichung City 40758, Taiwan, ROC
3
Department of Forensic Science, Central Police University, 56 Shu-Jen Road, Kwei-San, Taoyuan 33304, Taiwan, ROC
4
Department of Forensic Medicine, College of Medicine, National Taiwan University, No. 1 Jen-Ai Road Section 1, Taipei 10051, Taiwan, ROC
5
Forensic Biology Division, Criminal Investigation Bureau, National Police Agency, No. 5 Lane 553, Sec. 4, Zhongxiao E. RD., Xinyi District, Taipei 11072, Taiwan, ROC
6
School of Biological Sciences, Flinders University, Adelaide 5001, Australia
and a gender marker, were combined into a 10-plex methylation-specific PCR single-base-extension (MSPSBE) system. Results Analysis of 65 DNA samples isolated from venous blood, semen, vaginal fluid, saliva, and menstrual blood that had been treated with bisulfite, resulted in all eight markers detecting the body fluid to which they were designed. Unambiguous body fluid identification occurred from both single sources of body fluids and complex mixtures. A threshold was devised for each marker to minimize the chance of a false inclusion. 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. Conclusions The results demonstrated the 10-plex MSPSBE system established in this study is both sensitive and specific when applied to body fluid identification and can be readily adopted into forensic practice. Keywords DNA methylatio
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