Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples

Silva, Deborah S.B.S.; Antunes, Joana; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice Sampaio; McCord, Bruce

doi:10.1016/j.fsigen.2016.01.017

Cited by 74 publications

(49 citation statements)

References 12 publications

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“…One method is to treat the DNA with sodium bisulfite as this changes un-methylated cytosine into uracil while methylated cytosine remains unaffected [14]. The methylation status of any PCR product can be determined by the presence of either thymine, representing a lack of methylation, or the presence of a cytosine, indicating methylation; this can be detected by pyrosequencing [15][16][17][18] or single-baseextension (SBE) [19][20][21][22]. Bisulfite conversion can lead to the undesired side-effect of DNA degradation [23], which is not ideal if the sample is low template in nature or of poor quality.…”

Section: Introductionmentioning

confidence: 99%

Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system

Lin

Tsai

Lee

et al. 2016

Forensic Science International: Genetics

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Section: Introductionmentioning

confidence: 99%

Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system

Lin

Tsai

Lee

et al. 2016

Forensic Science International: Genetics

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“…Therefore, amplifying other markers like the ones described in the work of Silva et al . (2016) may allow mixture deconvolution using pyrosequencing. However, the identification of more genome locations with better sensitivity would be useful.…”

Section: Discussionmentioning

confidence: 99%

Forensic discrimination of vaginal epithelia by DNA methylation analysis through pyrosequencing

et al. 2016

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The accurate identification of body fluids from crime scenes can aid in the discrimination between criminal and innocent intent. This research aimed to determine if the levels of DNA methylation in the locus PFN3A could be used to discriminate vaginal epithelia from other body fluids. In this work we bisulfite-modified and amplified DNA samples from blood, saliva, semen, and vaginal epithelia using primers for PFN3A. Through pyrosequencing we were able to show that vaginal epithelia present distinct methylation levels when compared to other body fluids. Mixtures of different body fluids present methylation values that correlate with single-source body fluid samples and the primers for PFN3A are specific for primates. This report successfully demonstrated that the analysis of methylation in the PFN3A locus can be used for vaginal epithelia discrimination in forensic samples.

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“…Of all molecular based techniques for forensic serology, epigenetics has emerged as a favorable procedure due to its easy integration into the forensic workflow. Standard extraction methods are used, and stored extracts can easily be evaluated years later for the status of methylation markers .…”

Section: Introductionmentioning

confidence: 99%

“…This modification to cytosine residues in DNA has proven to be a more stable source of information when compared to RNA and protein‐based assays . Tests have demonstrated that methylation levels are preserved in blood and semen for at least 20 years . It has also been demonstrated that DNA methylation levels at certain loci vary depending on the cell type .…”

Section: Introductionmentioning

confidence: 99%

Development of a body fluid identification multiplex via DNA methylation analysis

et al. 2019

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The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers – BCAS4, CG06379435, VE_8, and ZC3H12D – were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.

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Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples

Cited by 74 publications

References 12 publications

Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system

Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system

Forensic discrimination of vaginal epithelia by DNA methylation analysis through pyrosequencing

Development of a body fluid identification multiplex via DNA methylation analysis

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