Skip to main content
SpringerLink
Log in

Sequenzierung aus einzelnen Epithelzellen

HV1- und HV2-Region der mitochondrialen DNA

Sequencing from single epithelial cells

HV1 and HV2 regions of mitochondrial DNA

  • Originalien
  • Published:
Rechtsmedizin Aims and scope Submit manuscript

Zusammenfassung

Die geringste morphologisch abgrenzbare Einheit einer biologischen Spur ist eine einzelne Zelle. Dabei handelt es sich überwiegend um Epithelzellen durch Hautabschilferungen oder aus Sekreten. Mithilfe eines Mikromanipulators, wie er für die intrazytoplasmatische Spermieninjektion (ICSI) verwendet wird, und selbst hergestellten Pipetten wurden aus Ausstrichen Einzelzellen isoliert bzw. aufgenommen und anschließend die beiden hypervariablen Regionen (HV1 und HV2) der mitochondrialen (mt)DNA amplifiziert und sequenziert. Insgesamt wurden 175 einzelne Epithelzellen von 6 verschiedenen Probanden untersucht. Aus 57 Einzelzellen wurde eine „single polymerase chain reaction“ (Single-PCR) der HV1-Region durchgeführt und in 77% der PCR ein Amplifikat nachgewiesen. Aus 22 Einzelzellen gelang dies für die HV2-Region, mit Nachweis des Amplifikats in 90% der PCR. An weiteren 96 Einzelzellen wurde eine gemeinsame Amplifikation von HV1- und HV2-Region in einem PCR-Ansatz vorgenommen; der Amplifikatnachweis lag bei 80% der PCR. Während aller Versuche waren keine Kontaminationen zu verzeichnen, d. h. die untersuchten Proben ergaben keine andere Sequenz als die erwartete und zeigten keine Mischspur. Die vorgestellte Methode kann in Fällen, in denen die „Short-tandem-repeat“- (STR-)Typisierung und konventionelle Sequenzierung der mtDNA nicht oder nur eingeschränkt möglich ist, Anwendung finden. Dies gilt insbesondere für Mischspuren und Untersuchungen zur Abschätzung von Heteroplasmien in der mtDNA.

Abstract

The smallest morphologically defined unit of biological trace evidence is a single cell. In most cases these are exfoliated epithelial cells or cells from secretions. With the aid of a micromanipulator, such as is used for intracytoplasmic sperm injections (ICSI) and self-made pipettes single cells were isolated, or lifted from smear preparations and both hypervariable regions (HV1 and HV2) of mitochondrial DNA (mtDNA) were subsequently amplified and sequenced. In total 175 single epithelial cells from 6 different subjects were examined. Single PCR for the HV1 region was performed for 57 of the single cells and PCR products could be demonstrated in 77% of cases. This procedure was also performed for the HV2 region from 22 single cells with PCR products being demonstrated in 90% of cases. For a further 96 single cells combined amplification of the HV1 and HV2 regions was performed in one PCR reaction and PCR products could be demonstrated in 80% of cases. During all experimental settings no contamination occurred meaning that none of the samples examined yielded any other sequence than the expected one and did not show a mixture of samples. The presented method can be applied to cases in which STR typing and conventional sequencing of mtDNA is limited or impossible. This particularly applies to mixed stains and examinations assessing heteroplasmy in mtDNA.

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

Abb. 1
Abb. 2
Abb. 3

Literatur

  1. Anderson S, Bankier AT, Barrell BG et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–465

    Article  CAS  PubMed  Google Scholar 

  2. Andréasson H, Nilsson M, Budowle B et al (2006) Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing. Int J Legal Med 120:183–390

    Article  Google Scholar 

  3. Andrews RM, Kubacka I, Chinnery PF et al (1999) Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 23(2):147

    Article  CAS  PubMed  Google Scholar 

  4. Anoruo B, Oorschot R van, Mitchell J, Howells D (2007) Isolating cells from non-sperm cellular mixtures using the PALM microlaser micro dissection system. Forensic Sci Int 173:93–96

    Article  CAS  PubMed  Google Scholar 

  5. Anslinger K, Bayer B, Mack B, Eisenmenger W (2007) Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling. Int J Legal Med 121:54–56

    Article  CAS  PubMed  Google Scholar 

  6. Bär W, Brinkmann B, Budowle B et al (2000) DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing. Int J Legal Med 113:193–196

    Article  PubMed  Google Scholar 

  7. Brandstätter A, Niederstätter H, Parson W (2004) Monitoring the inheritance of heteroplasmy by computer-assisted detection of mixed basecalls in the entire human mitochondrial DNA control region. Int J Legal Med 118:47–54

    Article  PubMed  Google Scholar 

  8. Brandstatter A, Parson W (2003) Mitochondrial DNA heteroplasmy or artefacts – a matter of the amplification strategy? Int J Legal Med 117:180–184

    Article  PubMed  Google Scholar 

  9. Brinkmann B (2004) Forensische DNA-Analytik. Dtsch Arztebl 101:A2329–A2335

    Google Scholar 

  10. Castella V, Dimo-Simonin M, Brandt-Casadevall C et al (2006) Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers. Int J Legal Med 120:67–72

    Article  CAS  PubMed  Google Scholar 

  11. Dettmeyer R (2008) Virusinduzierte Myokarditis beim mutmaßlichen plötzlichen Kindstod: Neue Erkenntnisse durch immunhistochemische und molekularpathologische Untersuchungen. Rechtsmedizin 18:365–376

    Article  Google Scholar 

  12. Di Martino D, Giuffrè G, Staiti N et al (2004) Single sperm cell isolation by laser microdissection. Forensic Sci Int 146:S151–S153

    Article  Google Scholar 

  13. Dimo-Simonoin N, Grange F, Taroni F et al (2000) Forensic evaluation of mtDNA in a population from south west Switzerland. Int J Legal Med 113:89–97

    Article  Google Scholar 

  14. Elliot K, Hill DS, Lambert C et al (2003) Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides. Forensic Sci Int 137:28–36

    Article  Google Scholar 

  15. Findlay I (2003) DNA identification from extreme samples such as single cells and difficult samples: uses including forensics and counter-terrorist applications. Oral Presentation: International Symposium on Forensic DNA Technologies, Münster (Germany), 19.–20.09.2003, Abstract (IP-2). Rechtsmedizin 13:273–274

    Google Scholar 

  16. Findlay I, Taylor A, Quirke P et al (1997) DNA fingerprinting from single cells. Nature 389:555–556

    Article  CAS  PubMed  Google Scholar 

  17. Garvin AM, Holzgreve W, Hahn S (1998) Highly accurate analysis of heterozygous loci by single cell PCR. Nucleic Acids Res 26:3468–3472

    Article  CAS  PubMed  Google Scholar 

  18. He L, Chinnery PF, Durham SE et al (2002) Detection and qualification of mitochondrial DNA deletions in individual cells by real-time PCR. Nucleic Acids Res 30(14):e68

    Article  PubMed  Google Scholar 

  19. Holland MM, Fisher DL, Mitchell LG et al (1993) Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War. J Forensic Sci 38:542–553

    CAS  PubMed  Google Scholar 

  20. Höss M, Kohn M, Pääbo S (1992) Excrement analysis by PCR. Nature 359:199

    Article  PubMed  Google Scholar 

  21. 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 121:184–191

    Article  PubMed  Google Scholar 

  22. Klein CA, Seidl S, Petat-Dutter K et al (2002) Combined transcriptome and genome analysis of single micrometastatic cells. Nat Biotechnol 20:387–392

    Article  CAS  PubMed  Google Scholar 

  23. Kramer N, Heidorn F, Weiler G, Verhoff MA (2003) Heteroplasmie der mtDNA bei einem zweieiigen Zwillingspaar. Arch Kriminol 211:98–105

    PubMed  Google Scholar 

  24. Lutz S, Weisser HJ, Heizmann J, Pollak S (1996) MtDNA as a tool for identification of human remains; identification using mtDNA. Int J Legal Med 109:205–209

    Article  CAS  PubMed  Google Scholar 

  25. Lutz S, Weisser HJ, Heizmann J, Pollak S (2000) Mitochondrial heteroplasmy among maternally related individuals. Int J Legal Med 113:155–161

    Article  CAS  PubMed  Google Scholar 

  26. Lutz-Bonengel S, Sänger T, Heinrich M et al (2007) Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip. Int J Legal Med 121:68–73

    Article  PubMed  Google Scholar 

  27. Lutz-Bonengel S, Sänger T, Parson W et al (2008) Single lymphocytes from two healthy individuals with mitochondrial point heteroplasmy are mainly homoplasmatic. Int J Legal Med 122:189–197

    Article  PubMed  Google Scholar 

  28. Malik S, Sodoyo H, Praqmoonjago P et al (2002) Evidence for the de novo regeneration of the pattern of length heteroplasmy associated with the T16189C variant in the control (D-loop) region of mitochondrial DNA. J Hum Genet 47:122–130

    Article  CAS  PubMed  Google Scholar 

  29. Melton T, Nelson K (2001) Forensic mitochondrial DNA analysis. Two years of commercial casework experiences in the United States. Croat Med J 42:298–303

    CAS  PubMed  Google Scholar 

  30. Morley JM, Bark JE, Evans CE et al (1999) Validation mitochondrial DNA minisequencing for forensic casework. Int J Legal Med 112:241–248

    Article  CAS  PubMed  Google Scholar 

  31. Murray C, McAlister C, Elliot K (2007) Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault. Forensic Sci Int Genet 1:247–252

    Article  PubMed  Google Scholar 

  32. Parson W (2009) Bedeutung der mtDNA-Analyse für forensische Fragestellungen. Rechtsmedizin 19:183–194

    Article  Google Scholar 

  33. Parson W, Brandstatter A, Alonso A et al (2004) The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives. Forensic Sci Int 139:215–226

    Article  CAS  PubMed  Google Scholar 

  34. Parsons TJ, Muniec DS, Sullivan K et al (1997) A high observed substitution rate in the human mitochondrial DNA control region. Nat Genet 15:363–368

    Article  CAS  PubMed  Google Scholar 

  35. Ray PF, Vekemans M, Munnich A (2001) Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination. Mol Hum Reprod 7:489–494

    Article  CAS  PubMed  Google Scholar 

  36. Reuss E, Kauferstein S, Zehner R et al (2008) Sicherung und Auswertung von latenten DNA-Spuren im Bereich der Eigentumskriminalität – Ein Feldversuch. Rechtsmedizin 18:251–256

    Article  Google Scholar 

  37. Sanders CT, Sanchez N, Ballantyne J, Peterson DA (2006) Laser microdissection separation of pure spermatozoa. J Forensic Sci 51:748–757

    Article  CAS  PubMed  Google Scholar 

  38. Strom CM, Rechitsky S (1998) Use of nested PCR to identify charred human remains and minute amounts of blood. J Forensic Sci 43(3):696–700

    CAS  PubMed  Google Scholar 

  39. Szibor R, Plate I, Heinrich M et al (2007) Mitochondrial D-loop (CA)n repeat length heteroplasmy: frequency in a German population sample and inheritance studies in two pedigrees. Int J Legal Med 121:207–213

    Article  PubMed  Google Scholar 

  40. Szibor R, Plate I, Schmitter H et al (2006) Forensic mass screening using mtDNA. Int J Legal Med 120:372–376

    Article  PubMed  Google Scholar 

  41. Tully G, Bär W, Brinkmann B et al (2001) Consideration by the European DNA profiling (EDNAP) group on the working practices, nomenclatures and interpretation of mitochondrial DNA profiles. Forensic Sci Int 124:83–91

    Article  CAS  PubMed  Google Scholar 

  42. Vandewoestyne M, Van Hoofstat D, Van Nieuwerburgh F, Deforce D (2009) Automatic detection of spermatozoa for laser capture microdissection. Int J Legal Med 123:169–175

    Article  PubMed  Google Scholar 

  43. Verhoff MA, Heidorn F, Oehmke S, Weiler G (2002) Beitrag zur Problematik der DNA-Typisierung von Kot. Rechtsmedizin 12:172–174

    Article  Google Scholar 

  44. Von Eggeling F, Michel S, Günther M et al (1997) Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphisms. Hum Genet 99:266–270

    Article  Google Scholar 

  45. Widjojoatmodjo MN, Fluit AC, Torensma R et al (1992) The magnetic immunopolymerase chain reaction assay for direct detection of salmonellae in faecal samples. J Clin Microbiol 30:3195–3199

    CAS  PubMed  Google Scholar 

  46. Wilson MR, DiZinno JA, Polanskey D et al (1995) Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med 18:68–74

    Article  Google Scholar 

Download references

Interessenkonflikt

Keine Angaben

Author information

Authors and Affiliations

  1. Institut für Rechtsmedizin, Justus-Liebig-Universität Gießen, Frankfurter Str. 58, 35392, Gießen, Deutschland

    S. Brück, V. Thias, F. Heidorn, C. Gruber, N. Kramer, H. Evers &  M.A. Verhoff

Authors
  1. S. Brück
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. Thias
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F. Heidorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Gruber
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N. Kramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. H. Evers
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M.A. Verhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M.A. Verhoff.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brück, S., Thias, V., Heidorn, F. et al. Sequenzierung aus einzelnen Epithelzellen. Rechtsmedizin 20, 25–33 (2010). https://doi.org/10.1007/s00194-009-0649-5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00194-009-0649-5

Schlüsselwörter

Keywords

Search

Navigation