Skip to main content
SpringerLink
Log in

High resolution melt curve analysis based on methylation status for human semen identification

  • Technical Report
  • Published:
Forensic Science, Medicine, and Pathology Aims and scope Submit manuscript

Abstract

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

References

  1. Vidaki A, Daniel B, Court DS. Forensic DNA methylation profiling-potential opportunities and challenges. Forensic Sci Int Genet. 2013;7:499–507.

    Article  CAS  PubMed  Google Scholar 

  2. Madi T, Balamurugan K, Bombardi R, Duncan G, McCord B. The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfate modification and pyrosequencing. Electrophoresis. 2012;33:1736–45.

    Article  CAS  PubMed  Google Scholar 

  3. Lee HY, Park MJ, Choi A, An JH, Yang WI, Shin KJ. Potential forensic application of DNA methylation profiling to body fluid identification. Int J Legal Med. 2011;126:55–62.

    Article  PubMed  Google Scholar 

  4. An JH, Choi A, Shin KJ, Yang WI, Lee HY. DNA methylation-specific multiplex assays for body fluid identification. Int J Legal Med. 2013;127:35–43.

    Article  PubMed  Google Scholar 

  5. Watanabe K, Akutsu T, Sakurada K. Development of a real-time PCR-based method for analyzing semen-specific unmethylated DNA regions and methylation status in aged body fluid stains. J Forensic Sci. 2016;61:S208–12.

    Article  CAS  PubMed  Google Scholar 

  6. Choi A, Shin KJ, Yang WI, Lee HY. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA. Int J Legal Med. 2014;128:33–41.

    Article  PubMed  Google Scholar 

  7. Frumkin D, Wasserstrom A, Budowle B, Davidson A. DNA methylation-based forensic tissue identification. Forensic Sci Int Genet. 2011;5:517–24.

    Article  CAS  PubMed  Google Scholar 

  8. Lopez CMR, Croxford AE, Wilkinson MJ. High-resolution melt analysis for SNP discovery, linkage mapping and analysis of DNA methylation. Comp Biochem Physiol A Physiol. 2008;150:S49–50.

    Article  Google Scholar 

  9. Malentacchi F, Forni G, Vinci S, Orlando C. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol. Nucleic Acids Res. 2009;37:e86.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Smith E, Jones ME, Drew PA. Quantitation of DNA methylation by melt curve analysis. BMC Cancer. 2008;9:123.

    Article  Google Scholar 

  11. Hanson E, Ballantyne J. Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification. Forensic Sci Int Genet Suppl Ser. 2013;4:e125–6.

    Article  Google Scholar 

  12. Hanson EK, Ballantyne J. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex high resolution melt (HRM) analysis. F1000 Res. 2014;2:281.

    Google Scholar 

  13. Antunes J, Silva DS, Balamurugan K, Duncan G, Alho CS, McCord B. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains. Anal Biochem. 2016;494:40–5.

    Article  CAS  PubMed  Google Scholar 

  14. Walsh S, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques. 1991;10:506–13.

    CAS  PubMed  Google Scholar 

  15. Moore D, Dowhan D. Purification and concentration of DNA from aqueous solutions. In: Ausubel F, Brent R, Kingston R, Moore D, Seidman JG, Smith J, Struhl K, editors. Short protocols in molecular biology. New Jersey: John Wiley and Sons; 2003 .Unit 2.1A

    Google Scholar 

  16. Instruction manual EZ DNA-Methylation Lightning Kit™. Irvine, CA: Zymo Research. Microcon Centrifugal Filter Devices user guide. Billerica, MA: EMD Millipore Corporation; 2013.

  17. http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi. Accessed 6 Oct 2014.

  18. Epitect® HRM™ PCR Handbook. Hilden: Qiagen Corporation; 2009.

  19. Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A. 2002;99:3740–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Larsen F, Gundersen F, Lopez R, Prydz H. CpG islands as gene markers in the human genome. Genomics. 1992;13:1095–107.

    Article  CAS  PubMed  Google Scholar 

  21. Weidner CI, Lin Q, Koch CM, Eisele L, Beier F, Ziegler P, et al. Aging of blood can be tracked by DNA methylation changes at three CpG sites. Genome Biol. 2014;15:R24.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Forensic Science Program, Cedar Crest College, 100 College Drive, Allentown, PA, 18104, USA

    Caitlyn Fachet, Lawrence Quarino & K. Joy Karnas

Authors
  1. Caitlyn Fachet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lawrence Quarino
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Joy Karnas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lawrence Quarino.

Ethics declarations

Funding

This study was funded by the Forensic Science Program at Cedar Crest College.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

The Institutional Review Board of Cedar Crest College approved this project for use with human subjects. Approval for use of human subjects was granted only after information regarding volunteer recruitment procedures, methods of sample collection, and assurances of anonymity and confidentiality were adequately described to the Institutional Review Board of Cedar Crest College. In addition, the Institutional Review Board ensured that any possible risks associated with the collection of samples in this study were explained to the volunteers and that they had a right to withdraw from the study at any time. The names of human subjects used in this study are known only to author Fachet.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fachet, C., Quarino, L. & Karnas, K.J. High resolution melt curve analysis based on methylation status for human semen identification. Forensic Sci Med Pathol 13, 86–91 (2017). https://doi.org/10.1007/s12024-016-9825-6

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12024-016-9825-6

Keywords

Search

Navigation