International Journal of Legal Medicine

, Volume 123, Issue 2, pp 177–184

Forensic species identification based on size variation of mitochondrial DNA hypervariable regions

  • Hiroaki Nakamura
  • Tomonori Muro
  • Shinji Imamura
  • Isao Yuasa
Technical Note

Abstract

In this study, two new systems for species identification were developed based on size variation of mitochondrial DNA hypervariable regions among animals: one was a conventional method using non-fluorescent primer sets and agarose gel electrophoresis and the other was an automatic method using fluorescent primer sets and capillary electrophoresis. DNA samples from 18 mammal, four birds, and 19 fish species were amplified using three primer sets specific for mammals, birds, and fishes, respectively. The differences in the sizes of the polymerase chain reaction (PCR) products, ranging from about 350 to 900 bp, permitted us to identify species. These systems were successfully applied to various specimens from several criminal cases. In unknown samples, which were different in size from reference DNA markers, sequencing of the PCR products and subsequent BLAST analysis helped to identify species. Furthermore, the sequence data provided us with information on individuals. Because these species identification methods are very simple, easy, rapid, and exact, they are useful in the field of forensic science.

Keywords

Capillary electrophoresis Hypervariable regions Mitochondrial DNA Species identification Universal primer 

Supplementary material

414_2008_306_MOESM1_ESM.doc (36 kb)
Supplementary Table S1List of animals investigated in this study and the product size ± threshold value used for automated species calling (DOC 35.5 KB)
414_2008_306_MOESM2_ESM.doc (36 kb)
Supplementary Table S2Nucleotide sequences of priming position and the size of product estimated from the sequences of animal D-loops (DOC 36.5 KB)
414_2008_306_MOESM3_ESM.xls (26 kb)
Supplementary Table S3Haplotypes and sequences of mtDNA-HV in 82 dogs (XLS 25.5 KB)
414_2008_306_MOESM4_ESM.xls (36 kb)
Supplementary Table S4Haplotypes and sequences of mtDNA-HV in 37 cats (XLS 36.5 KB)

References

  1. 1.
    Bataille M, Crainic K, Leterreux M, Durigon M, Mazancourt de P (1999) Multiplex amplification of mitochondrial DNA for human and species identification in forensic evaluation. Forensic Sci Int 99:165–170PubMedCrossRefGoogle Scholar
  2. 2.
    Parson W, Pegoraro K, Niederstätter H, Föger M, Steinlechner M (2000) Species identification by means of the cytochrome b gene. Int J Legal Med 114:23–28PubMedCrossRefGoogle Scholar
  3. 3.
    Branicki W, Kupiec T, Pawlowski R (2003) Validation of cytochrome b sequence analysis as a method of species identification. J Forensic Sci 48:83–87PubMedGoogle Scholar
  4. 4.
    Bravi CM, Lirón JP, Mirol PM, Ripoli MV, Peral-García P, Giovambattista G (2004) A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene. Legal Med 6:246–251PubMedCrossRefGoogle Scholar
  5. 5.
    An J, Lee MY, Min MS, Lee MH, Lee H (2007) A molecular genetic approach for species identification of mammals and sex determination of birds in a forensic case of poaching from South Korea. Forensic Sci Int 167:59–61PubMedCrossRefGoogle Scholar
  6. 6.
    Linacre A, Lee JC (2005) Species determination: the role and use of the cytochrome b gene. In: Carracedo A (ed) Forensic DNA typing protocols. Humana, Totowa, pp 45–51Google Scholar
  7. 7.
    Wetton JH, Tsang CS, Roney CA, Spriggs AC (2002) An extremely sensitive species-specific ARMS PCR test for the presence of tiger bone DNA. Forensic Sci Int 126:137–144PubMedCrossRefGoogle Scholar
  8. 8.
    Lee JC, Hsieh HM, Huang LH et al (2008) Ivory identification by DNA profiling of cytochrome b gene. Int J Legal Med. doi:10.1007/s00414-008-0264-0
  9. 9.
    Wells JD, Wall R, Stevens JR (2007) Phylogenetic analysis of forensically important Lucilia flies based on cytochrome oxidase I sequence: a cautionary tale for forensic species determination. Int J Legal Med 121:229–233PubMedCrossRefGoogle Scholar
  10. 10.
    Balitzki-Korte B, Anslinger K, Bartsch C, Rolf B (2005) Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene. Int J Legal Med 119:291–294PubMedCrossRefGoogle Scholar
  11. 11.
    Melton T, Holland C (2007) Routine forensic use of the mitochondrial 12S ribosomal RNA gene for species identification. J Forensic Sci 52:1305–1307PubMedGoogle Scholar
  12. 12.
    Kitano T, Umetsu K, Tian W, Osawa M (2007) Two universal primer sets for species identification among vertebrates. Int J Legal Med 121:423–427PubMedCrossRefGoogle Scholar
  13. 13.
    Imaizumi K, Akutsu T, Miyasaka S, Yoshino M (2007) Development of species identification tests targeting the 16S ribosomal RNA coding region in mitochondrial DNA. Int J Legal Med 121:184–191PubMedCrossRefGoogle Scholar
  14. 14.
    Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci U S A 86:6196–6200PubMedCrossRefGoogle Scholar
  15. 15.
    Murray BW, McClymont RA, Strobeck C (1995) Forensic identification of ungulate species using restriction digests of PCR-amplified mitochondrial DNA. J Forensic Sci 40:943–951PubMedGoogle Scholar
  16. 16.
    Wu H, Wan QH, Fang SG, Zhang SY (2005) Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies. Forensic Sci Int 148:101–105PubMedCrossRefGoogle Scholar
  17. 17.
    Tobe SS, Linacre AM (2008) A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene. Electrophoresis 29:340–347PubMedCrossRefGoogle Scholar
  18. 18.
    Bellis C, Ashton KJ, Freney L, Blair B, Griffiths LR (2003) A molecular genetic approach for forensic animal species identification. Forensic Sci Int 134:99–108PubMedCrossRefGoogle Scholar
  19. 19.
    Naito E, Dewa K, Yamanouchi H, Kominami R (1992) Ribosomal ribonucleic acid (rRNA) gene typing for species identification. J Forensic Sci 37:396–403PubMedGoogle Scholar
  20. 20.
    Lopez JV, Cevario S, O’Brien SJ (1996) Complete nucleotide sequences of the domestic cat (Felis catus) mitochondrial genome and a transposed mtDNA tandem repeat (Numt) in the nuclear genome. Genomics 33:229–246PubMedCrossRefGoogle Scholar
  21. 21.
    Buroker NE, Brown JR, Gilbert TA, O’Hara PJ, Beckenbach AT, Thomas WK, Smith MJ (1990) Length heteroplasmy of sturgeon mitochondrial DNA: an illegitimate elongation model. Genetics 124:157–163PubMedGoogle Scholar
  22. 22.
    Wilkinson GS, Chapman AM (1991) Length and sequence variation in evening bat D-loop mtDNA. Genetics 128:607–617PubMedGoogle Scholar
  23. 23.
    Ishiguro N, Nakajima A, Horiuchi M, Shinagawa M (2002) Multiple nuclear pseudogenes of mitochondrial DNA exist in the canine genome. Mam Genome 13:365–372CrossRefGoogle Scholar
  24. 24.
    Allen M, Engström AS, Meyers S et al (1998) Mitochondrial DNA sequencing of shed hairs and saliva on robbery caps: sensitivity and matching probabilities. J Forensic Sci 43:453–464PubMedGoogle Scholar
  25. 25.
    Gabriel MN, Huffine EF, Ryan JH, Holland MM, Parsons TJ (2001) Improved mtDNA sequence analysis of forensic remains using a “mini-primer set” amplification strategy. J Forensic Sci 46:247–253PubMedGoogle Scholar
  26. 26.
    Nelson K, Melton T (2007) Forensic mitochondrial DNA analysis of 116 casework skeletal samples. J Forensic Sci 52:557–561PubMedCrossRefGoogle Scholar
  27. 27.
    Vilá C, Savolainen P, Maldonado JE et al (1997) Multiple and ancient origins of the domestic dog. Science 276:1687–1689PubMedCrossRefGoogle Scholar
  28. 28.
    Savolainen P, Rosén B, Holmberg A, Leitner T, Uhlén M, Lundeberg J (1997) Sequence analysis of domestic dog mitochondrial DNA for forensic use. J Forensic Sci 42:593–600PubMedGoogle Scholar
  29. 29.
    Okumura N, Ishiguro N, Nakano M, Matsui A, Shigehara N, Nishimoto T, Sahara M (1999) Variations in mitochondrial DNA of dogs isolated from archaeological sites in Japan and neighbouring islands. Anthrop Sci 107:213–228Google Scholar
  30. 30.
    Pereira L, Asch BV, Amorim A (2004) Standardisation of nomenclature for dog mtDNA D-loop: a prerequisite for launching a Canis familiaris database. Forensic Sci Int 141:99–108PubMedCrossRefGoogle Scholar
  31. 31.
    Angleby H, Savolainen P (2005) Forensic informativity of domestic dog mtDNA control region sequences. Forensic Sci Int 154:99–110PubMedCrossRefGoogle Scholar
  32. 32.
    Eichmann C, Parson W (2007) Molecular characterization of the canine mitochondrial DNA control region for forensic applications. Int J Legal Med 121:411–416PubMedCrossRefGoogle Scholar
  33. 33.
    Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  34. 34.
    Nakahara H, Sekiguchi K, Imaizumi K, Mizuno N, Kasai K (2008) Heteroplasmies detected in an amplified mitochondrial DNA control region from a small amount of template. J Forensic Sci 53:306–311PubMedCrossRefGoogle Scholar
  35. 35.
    Schneider PM, Seo Y, Rittner C (1999) Forensic mtDNA hair analysis excludes a dog from having caused a traffic accident. Int J Legal Med 112:315–316PubMedCrossRefGoogle Scholar
  36. 36.
    D’Andrea F, Fridez F, Coquoz R (1998) Preliminary experiments on the transfer of animal hair during simulated criminal behavior. J Forensic Sci 43:1257–1258Google Scholar
  37. 37.
    Gupta SK, Thangaraj K, Singh L (2006) A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin. J Forensic Sci 51:805–807PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Hiroaki Nakamura
    • 1
  • Tomonori Muro
    • 1
    • 2
  • Shinji Imamura
    • 1
    • 2
  • Isao Yuasa
    • 3
  1. 1.Criminal Investigation LaboratoryShimane Prefectural Police HeadquartersMatsueJapan
  2. 2.Department of Legal Medicine, Faculty of MedicineShimane UniversityIzumoJapan
  3. 3.Division of Legal Medicine, Faculty of MedicineTottori UniversityYonagoJapan

Personalised recommendations