Skip to main content
SpringerLink
Log in

HRM and SNaPshot as alternative forensic SNP genotyping methods

  • Original Article
  • Published:
Forensic Science, Medicine and Pathology Aims and scope Submit manuscript

Abstract

Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.

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
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Budowle B, van Daal A. Forensically relevant SNP classes. BioTechniques. 2008;44(5):603–10.

    Article  CAS  PubMed  Google Scholar 

  2. Kayser M, de Knijff P. Improving human forensics through advances in genetics, genomics and molecular biology. Nature Rev Genet. 2011;12(3):179–92.

    Article  CAS  PubMed  Google Scholar 

  3. Mehta B, Daniel R, Phillips C, McNevin D. Forensically relevant SNaPshot® assays for human DNA SNP analysis: a review. Int J Legal Med. 2017;131(1):21–37.

    Article  PubMed  Google Scholar 

  4. Sanchez JJ, Phillips C, Børsting C, Balogh K, Bogus M, Fondevila M, et al. A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis. 2006;27(9):1713–24.

    Article  CAS  PubMed  Google Scholar 

  5. Phillips C, Salas A, Sánchez JJ, Fondevila M, Gómez-Tato A, Álvarez-Dios J, et al. Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs. Forensic Sci Int Genet. 2007;1(3–4):273–80.

    Article  CAS  PubMed  Google Scholar 

  6. Walsh S, Liu F, Ballantyne KN, Oven MV, Lao O, Kayser M. IrisPlex: a sensitive DNA tool for accurate prediction of blue and brown eye colour in the absence of ancestry information. Forensic Sci Int Genet. 2011;5:170–80.

    Article  CAS  PubMed  Google Scholar 

  7. Mehta B, Daniel R, McNevin D. High resolution melting (HRM) of forensically informative SNPs. Forensic Sci Int Genet Supp Ser. 2013;4(1):e376–7.

    Article  Google Scholar 

  8. Venables SJ, Mehta B, Daniel R, Walsh SJ, van Oorschot RAH, McNevin D. Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework. Electrophoresis. 2014;35(21–22):3036–43.

    Article  CAS  PubMed  Google Scholar 

  9. Reed GH, Kent JO, Wittwer CT. High resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics. 2007;8(6):597–608.

    Article  CAS  PubMed  Google Scholar 

  10. Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem. 2003;49(3):396–406.

    Article  CAS  PubMed  Google Scholar 

  11. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, et al. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem. 2004;50(7):1156–64.

    Article  CAS  PubMed  Google Scholar 

  12. Walsh S, Lindenbergh A, Zuniga SB, Sijenb T. Knijff Pd, Kayser M, et al. developmental validation of the IrisPlex system: determination of blue and brown iris colour for forensic intelligence. Forensic Sci Int Genet. 2011;5(5):464–71.

    Article  CAS  PubMed  Google Scholar 

  13. AppliedBiosystems. ABI PRISM® SNaPshot™ Multiplex Kit. P/N 4323357 Rev. B ed: Thermo Fisher Scientific. 2010;1–42.

  14. Walsh S, Chaitanya L, Clarisse L, Wirken L, Draus-Barini J, Kovatsi L, et al. Developmental validation of the HIrisPlex system: DNA-based eye and hair colour prediction for forensic and anthropological usage. Forensic Sci Int Genet. 2014;9:150–61.

    Article  CAS  PubMed  Google Scholar 

  15. Westen AA, Matai AS, Laros JF, Meiland HC, Jasper M, de Leeuw WJ, et al. Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples. Forensic Sci Int Genet. 2009;3(4):233–41.

    Article  CAS  PubMed  Google Scholar 

  16. Swen JJ, Baak-Pablo RF, Guchelaar H-J, van der Straaten T. Alternative methods to a TaqMan assay to detect a tri-allelic single nucleotide polymorphism rs757210 in the HNF1β gene. Clin Chem Lab Med. 2012;50(2):279–84.

    Article  CAS  Google Scholar 

  17. Fondevila M, Phillips C, Santos C, Freire Aradas A, Vallone PM, Butler JM, et al. Revision of the SNPforID 34-plex forensic ancestry test: assay enhancements, standard reference sample genotypes and extended population studies. Forensic Sci Int Genet. 2013;7(1):63–74.

    Article  CAS  PubMed  Google Scholar 

  18. Seipp MT, Durtschi JD, Voelkerding KV, Wittwer CT. Multiplex amplicon genotyping by high-resolution melting. J Biomol Tech. 2009;20(3):160–4.

    PubMed  PubMed Central  Google Scholar 

  19. Nicklas JA, Buel E. Improving the efficiency of DNA casework analysis through simple, effective, PCR-based screening methods. NIJ Grant#2005-DA-BX-K003. 2011;1–115.

  20. Derzelle S, Laroche S, Le Fleche P, Hauck Y, Thierry S, Vergnaud G, et al. Characterization of genetic diversity of bacillus anthracis in France by using high-resolution melting assays and multilocus variable-number tandem-repeat analysis. J Clin Microbiol. 2011;49(12):4286–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bosmali I, Ganopoulos I, Madesis P, Tsaftaris A. Microsatellite and DNA-barcode regions typing combined with high resolution melting (HRM) analysis for food forensic uses: a case study on lentils (Lens culinaris). Food Res Int. 2012;46(1):141–7.

    Article  CAS  Google Scholar 

  22. Ganopoulos I, Bazakos C, Madesis P, Kalaitzis P, Tsaftaris A. Barcode DNA high-resolution melting (bar-HRM) analysis as a novel close-tubed and accurate tool for olive oil forensic use. J Sci Food Agr. 2013;93(9):2281–6.

    Article  CAS  Google Scholar 

  23. Malewski T, Draber-Mońko A, Pomorski J, Łoś M, Bogdanowicz W. Identification of forensically important blowfly species (Diptera: Calliphoridae) by high-resolution melting PCR analysis. Int J Legal Med. 2010;124(4):277–85.

    Article  PubMed  Google Scholar 

  24. Sijen T. Molecular approaches for forensic cell type identification: on mRNA, miRNA, DNA methylation and microbial markers. Forensic Sci Int Genet. 2015;18:21–32.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge financial support from the Australian Research Council (LP110100121 - From genotype to phenotype: Molecular photofitting for criminal investigations).

Author information

Authors and Affiliations

  1. National Centre for Forensic Studies, Faculty of Education, Science, Technology & Mathematics, University of Canberra, ACT, Bruce, 2617, Australia

    Bhavik Mehta & Dennis McNevin

  2. Office of the Chief Forensic Scientist, Forensic Services Department, Victoria Police, 31 Forensic Drive, Macleod, VIC, 3085, Australia

    Runa Daniel

Authors
  1. Bhavik Mehta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Runa Daniel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dennis McNevin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bhavik Mehta.

Ethics declarations

Ethics approval

All procedures performed in this study involving human participants were in accordance with the ethical standards of the University of Canberra Human Ethics Committee (Project number 11–19) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(PDF 588 kb)

ESM 2

(PDF 19 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mehta, B., Daniel, R. & McNevin, D. HRM and SNaPshot as alternative forensic SNP genotyping methods. Forensic Sci Med Pathol 13, 293–301 (2017). https://doi.org/10.1007/s12024-017-9874-5

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12024-017-9874-5

Keywords

Search

Navigation