Skip to main content
SpringerLink
Log in

Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence

  • Original Article
  • Published:
International Journal of Legal Medicine Aims and scope Submit manuscript

Abstract

DNA typing from forensic evidence is commonly used to identify individuals. However, when the quantity of the forensic evidence is insufficient, successful identification using DNA typing is impossible. Such evidence may also contain DNA from bacteria that occur naturally on the skin. In this study, we aimed to establish a profiling method using terminal restriction fragment length polymorphisms (T-RFLPs) of the amplified bacterial 16S ribosomal RNA (rRNA) gene. First, the extraction and digestion processes were investigated, and the T-RFLP profiling method using the 16S rRNA gene amplicon was optimized. We then used this method to compare the profiles of bacterial flora from the hands of 12 different individuals. We found that the T-RFLP profiles from one person on different days displayed higher similarity than those between individuals. In a principal component analysis (PCA), T-RFLPs from each individual were closely clustered in 11 out of 12 cases. The clusters could be distinguished from each other, even when the samples were collected from different conditions. No major change of the profile was observed after six months except in two cases. When handprints on glass plates were compared, 11 of 12 individuals were assigned to a few clusters including the cluster corresponding to the correct individual. In conclusion, a method for reproducible T-RFLP profiling of bacteria from trace amounts of handprints was established. The profiles were obtained for particular individuals clustered in PCA and were experimentally separable from other individuals in most cases. This technique could provide useful information for narrowing down a suspect in a criminal investigation.

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. Raymond JJ, van Oorschot RAH, Gunn PR, Walsh SJ, Roux C (2009) Trace evidence characteristics of DNA: a preliminary investigation of the persistence of DNA at crime scenes. Forensic Sci Int Genet 4:26–33

    Article  CAS  PubMed  Google Scholar 

  2. van Oorschot RAH, Jones MK (1997) DNA fingerprints from fingerprints. Nature 387:767

    Article  PubMed  Google Scholar 

  3. Daly DJ, Murphy C, McDermott SD (2012) The transfer of touch DNA from hands to glass, fabric and wood. Forensic Sci Int Genet 6:41–46

    Article  CAS  PubMed  Google Scholar 

  4. Dass SC (2013) Fingerprint-based recognition. Int Stat Rev 81:175–187

    Article  Google Scholar 

  5. Horswell J, Cordiner SJ, Maas EW, Martin TM, Sutherland KBW, Speir TW, Nogales B, Osborn AM (2002) Forensic comparison of soils by bacterial community DNA profiling. J Forensic Sci 47:350–353

    CAS  PubMed  Google Scholar 

  6. Macdonald CA, Ang R, Cordiner SJ, Horswell J (2011) Discrimination of soils at regional and local levels using bacterial and fungal T-RFLP profiling. J Forensic Sci 56:61–69

    Article  PubMed  Google Scholar 

  7. Lenz EJ, Foran DR (2010) Bacterial profiling of soil using genus-specific markers and multidimensional scaling. J Forensic Sci 55:1437–1442

    Article  CAS  PubMed  Google Scholar 

  8. Nakanishi H, Kido A, Ohmori T, Takada A, Hara M, Adachi N, Saito K (2009) A novel method for the identification of saliva by detecting oral streptococci using PCR. Forensic Sci Int 183:20–23

    Article  CAS  PubMed  Google Scholar 

  9. Benschop CCG, Quaak FCA, Boon ME, Sijen T, Kuiper I (2012) Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context? Int J Legal Med 126:303–310

    Article  PubMed  Google Scholar 

  10. Goga H (2012) Comparison of bacterial DNA profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners. Int J Legal Med 126:815–823

    Article  PubMed  Google Scholar 

  11. Fierer N, Lauber CL, Zhou N, McDonald D, Costello EK, Knight R (2010) Forensic identification using skin bacterial communities. Proc Natl Acad Sci U S A 107:6477–6481

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC et al (2009) Topographical and temporal diversity of the human skin microbiome. Science 324:1190–1192

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Gao Z, Tseng CH, Pei Z, Blaser MJ (2007) Molecular analysis of human forearm superficial skin bacterial biota. Proc Natl Acad Sci U S A 104:2927–2932

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R (2009) Bacterial community variation in human body habitats across space and time. Science 326:1694–1697

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Head IM, Saunders JR, Pickup RW (1998) Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol 35:1–21

    Article  CAS  PubMed  Google Scholar 

  16. Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522

    PubMed Central  CAS  PubMed  Google Scholar 

  17. Smith CJ, Danilowicz BS, Clear AK, Costello FJ, Wilson B, Meijer WG (2005) T-Align, a web-based tool for comparison of multiple terminal restriction fragment length polymorphism profiles. FEMS Microbiol Ecol 54:375–380

    Article  CAS  PubMed  Google Scholar 

  18. Nakahara H, Fujii K, Mizuno N, Yoshida K, Kasai K (2007) Evaluations of DNA quantification methods for forensic biological samples. Jpn J Forensic Sci Tech 12:13–26

    Article  Google Scholar 

  19. Fierer N, Jackson JA, Vilgalys R, Jackson RB (2005) Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol 71:4117–4120

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Schütte UME, Abdo Z, Bent SJ, Shyu C, Williams CJ, Pierson JD, Forney LJ (2008) Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize micrbial communities. Appl Microbiol Biotechnol 80:365–380

    Article  PubMed  Google Scholar 

  21. Lukow T, Dunfield PF, Liesack W (2000) Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microbiol Ecol 32:241–247

    Article  CAS  Google Scholar 

  22. Rees GN, Baldwin DS, Watson GO, Perryman S, Nielsen DL (2004) Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate ststistics. Antonie Van Leeuwenhoek 86:339–347

    Article  PubMed  Google Scholar 

  23. Meyers MS, Foran DR (2008) Spatial and temporal influences on bacterial profiling of forensic soil samples. J Forensic Sci 53:652–660

    Article  PubMed  Google Scholar 

  24. Thalib L, Kitching RL, Bhatti MI (1999) Principal component analysis for grouped data—a case study. Environmetrics 10:565–574

    Article  Google Scholar 

  25. R Development Core Team. R: a language and environment for statistical computing. http://www.R-project.org

  26. Clement BG, Kehl LE, DeBord KL, Kitts CL (1998) Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J Microbiol Methods 31:135–142

    Article  CAS  Google Scholar 

  27. Engebretson JJ, Moyer CL (2003) Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism. Appl Environ Microbiol 69:4823–4829

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Nocker A, Burr M, Camper AK (2007) Genotypic microbial community profiling: a critical technical review. Microb Ecol 54:276–289

    Article  CAS  PubMed  Google Scholar 

  29. Benschop CCG, van der Beek CP, Meiland HC, van Gorp AGM, Westen AA, Sijen T (2011) Low template STR typing: effect of replicate number and consensus method on genotyping reliability and DNA database search results. Forensic Sci Int Genet 5:316–328

    Article  CAS  PubMed  Google Scholar 

  30. Fierer N, Hamady M, Lauber CL, Knight R (2008) The influence of sex, handedness, and washing on the diversity of hand surface bacteria. Proc Natl Acad Sci U S A 105:17994–17999

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Maiuta ND, Schwarzentruber P (2011) Molecular detection of bacteria in calcium carbonate powder used in cosmetic formulations. Int J Cosmetic Sci 33:426–431

    Article  Google Scholar 

  32. Talan DA, Citron DM, Abrahamian FM, Moran GJ, Goldstein EJC (1999) Bacteriologic analysis of infected dog and cat bites. N Engl J Med 340:85–92

    Article  CAS  PubMed  Google Scholar 

  33. Flores GE, Bates ST, Knights D, Lauber CL, Stombaugh J, Knight R, Fierer N (2011) Microbial biogeography of public restroom surfaces. PLoS One 6:e28132

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Shyu C, Soule T, Bent SJ, Foster JA, Forney LJ (2007) MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes. Microb Ecol 53:562–570

    Article  CAS  PubMed  Google Scholar 

  35. Grice EA, Kong HH, Renaud G, Young AC, NISC comparative sequencing program, Bouffard GG, Blakesley RW, Wolfsberg TG, Turner ML, Segre JA (2008) A diversity profile of the human skin microbiota. Genome Res 18:1043–1050

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Marsh TL, Saxman P, Cole J, Tiedje J (2000) Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl Environ Microbiol 66:3616–3620

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Macdonald LM, Singh BK, Thomas N, Brewer MJ, Campbell CD, Dawson LA (2008) Microbial DNA profiling by multiplex terminal restriction fragment length polymorphism for forensic comparison of soil and the influence of sample condition. J Appl Microbiol 105:813–821

    Article  CAS  PubMed  Google Scholar 

  38. Horz HP, Rotthauwe JH, Lukow T, Liesack W (2000) Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products. J Microbiol Meth 39:197–204

    Article  CAS  Google Scholar 

  39. Lueders T, Friedrich MW (2003) Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microbiol 69:320–326

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Pérez-Jiménez JR, Kerkhof LJ (2005) Phylogeography of sulfate-reducing bacteria among disturbed sediments, disclosed by analysis of the dissimilatory sulfite reductase genes (dsrAB). Appl Environ Microbiol 71:1004–1011

    Article  PubMed Central  PubMed  Google Scholar 

  41. Rösch C, Bothe H (2005) Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Appl Environ Microbiol 71:2026–2035

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

We thank for all of the volunteers who provided bacterial samples for this study. We appreciate Mr. Yuta Tamaki (Department of Criminal Investigation, Oita Prefectural Police HQ) for his helpful discussions on the work in this paper.

Author information

Authors and Affiliations

  1. Laboratory of Soil Microbiology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan

    Eiji Nishi, Yukihiro Tashiro & Kenji Sakai

  2. Forensic Science Division, Department of Criminal Investigation, Oita Prefectural Police HQ, 3-1-1 Otemachi, Oita, 870-8502, Japan

    Eiji Nishi

Authors
  1. Eiji Nishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yukihiro Tashiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kenji Sakai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kenji Sakai.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PPT 178 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nishi, E., Tashiro, Y. & Sakai, K. Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence. Int J Legal Med 129, 425–433 (2015). https://doi.org/10.1007/s00414-014-1092-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00414-014-1092-z

Keywords

Search

Navigation