Advertisement

International Journal of Legal Medicine

, Volume 133, Issue 2, pp 411–417 | Cite as

Development of the MitoQ assay as a real-time quantification of mitochondrial DNA in degraded samples

  • Ka Tak WaiEmail author
  • Peter Gunn
  • Mark Barash
Original Article

Abstract

Mitochondrial DNA is a reliable genetic material for estimating maternally related haplogroups and ancestries. Exploring maternal DNA inheritance is particularly useful when nuclear DNA is degraded or limited, as the copy number of mitochondrial DNA is far greater than the copy number of nuclear DNA. Normal mitochondrial DNA copy number has been estimated to 100 copies per buccal epithelial cell, 4000 copies in skeletal cells and 7000 copies in myocardial cells. This estimation is usually performed via extrapolation from the nuclear DNA quantitation. It is essential to reduce this variability and accurately quantify the exact number of copies of mitochondrial DNA, especially in compromised samples of a forensic or ancient nature. While useful, the testing of mitochondrial DNA is often long and costly and comes with limited success. The accurate quantification of mitochondrial DNA using specific quantitative PCR assays can be used to make better decisions on the downstream testing and success of amplification. As a result, this study develops a real-time assay for the quantification of mitochondrial DNA copy number and assesses its performance on a set of degraded DNA samples. The developed MitoQ assay has been shown to be highly specific to the human mitochondrial genome with no amplification of nuclear pseudogenes being observed and outperformed a previously published concordant assay. Additionally, a high sensitivity was measured to 280 copies of mitochondrial DNA. Minimal variation was observed between each replication cycle, indicating the assay to be robust and repeatable. Overall, this study presents a real-time assay that is sensitive and robust to quantifying mitochondrial DNA copy number in degraded samples. Furthermore, there is potential to incorporate the assay as an additional target in current qPCR assays which use a six-dye chemistry and provide a complete overview of a sample’s quality and quantity.

Keywords

DNA quantification Mitochondrial DNA Forensic assay Copy number Degraded DNA Real-time PCR 

Notes

Acknowledgements

This research is supported by an Australian Government Training Program Scholarship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

414_2018_1956_MOESM1_ESM.pdf (154 kb)
ESM 1 (PDF 153 kb)
414_2018_1956_MOESM2_ESM.pdf (358 kb)
ESM 2 (PDF 358 kb)
414_2018_1956_MOESM3_ESM.pdf (23 kb)
ESM 3 (PDF 23 kb)
414_2018_1956_MOESM4_ESM.pdf (22 kb)
ESM 4 (PDF 21 kb)

References

  1. 1.
    Robin JD, Ludlow AT, LaRanger R, Wright WE, Shay JW (2016) Comparison of DNA quantification methods for next generation sequencing. Sci Rep 6:24067Google Scholar
  2. 2.
    Wieczorek P (2017) To NanoDrop®or not to NanoDrop®: choosing the most appropriate method for nucleic acid quantitation. Promega Corporation, WisconsinGoogle Scholar
  3. 3.
    Satoh M, Kuroiwa T (1991) Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp Cell Res 196(1):137–140Google Scholar
  4. 4.
    Tobe SS, Linacre AM (2008) A technique for the quantification of human and non-human mammalian mitochondrial DNA copy number in forensic and other mixtures. Forensic Sci Int Genet 2(4):249–256Google Scholar
  5. 5.
    Malik AN, Shahni R, Rodriguez-de-Ledesma A, Laftah A, Cunningham P (2011) Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias. Biochem Biophys Res Commun 412(1):1–7Google Scholar
  6. 6.
    Kavlick MF, Lawrence HS, Merritt RT, Fisher C, Isenberg A, Robertson JM, Budowle B (2011) Quantification of human mitochondrial DNA using synthesized DNA standards. J Forensic Sci 56(6):1457–1463Google Scholar
  7. 7.
    Andréasson H, Nilsson M, Budowle B, Lundberg H, Allen M (2006) Nuclear and mitochondrial DNA quantification of various forensic materials. Forensic Sci Int 164(1):56–64Google Scholar
  8. 8.
    Lopez JV, Yuhki N, Masuda R, Modi W, O’Brien SJ (1994) Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat. J Mol Evol 39(2):174–190Google Scholar
  9. 9.
    Ramos A, Barbena E, Mateiu L, del Mar González M, Mairal Q, Lima M, Montiel R, Aluja MP, Santos C (2011) Nuclear insertions of mitochondrial origin: database updating and usefulness in cancer studies. Mitochondrion 11(6):946–953Google Scholar
  10. 10.
    Tourmen Y, Baris O, Dessen P, Jacques C, Malthièry Y, Reynier P (2002) Structure and chromosomal distribution of human mitochondrial pseudogenes. Genomics 80(1):71–77Google Scholar
  11. 11.
    Walker JA, Hedges DJ, Perodeau BP, Landry KE, Stoilova N, Laborde ME, Shewale J, Sinha SK, Batzer MA (2005) Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification. Anal Biochem 337(1):89–97Google Scholar
  12. 12.
    Ramos A, Santos C, Alvarez L, Nogués R, Aluja MP (2009) Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification. Electrophoresis 30(9):1587–1593Google Scholar
  13. 13.
    Qiagen (2016) QIAamp® DNA mini and blood mini handbook. Qiagen, HildenGoogle Scholar
  14. 14.
    Wai KT, Barash M, Gunn P (2018) Performance of the early access AmpliSeq™ mitochondrial panel with degraded DNA samples using the ion torrent™ platform. Electrophoresis.  https://doi.org/10.1002/elps.201700371
  15. 15.
    Thermo Fisher Scientific (2017) Quantifiler™ HP and trio DNA quantification kits. Thermo Fisher Scientific, CaliforniaGoogle Scholar
  16. 16.
    Qiagen (2010) HotStarTaq® plus PCR handbook. Qiagen, HildenGoogle Scholar
  17. 17.
    Anderson S, Bankier A, de Bruijn M, Coulson AR, Drouin J, Eperon I, Nierlich D, Roe B, Sanger F, Schreier P, Smith A, Staden R, Young I (1981) Sequence and organization of the human mitochondrial genome. Nature 290(5806):457–465Google Scholar
  18. 18.
    Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N (1999) Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 23(2):147–147Google Scholar
  19. 19.
    Bio-Rad Laboratories (2014) PrimePCR™ assays, panels, and controls for real-time PCR. Bio-Rad Laboratories, CaliforniaGoogle Scholar
  20. 20.
    Alonso A, Martí P, Albarrán C, Garcí P, Garcí O, de Simón LF, Garcí J, Sancho M, de la Rúa C, Fernández-Piqueras J (2004) Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies. Forensic Sci Int 139(2):141–149Google Scholar
  21. 21.
    Ellison SL, English CA, Burns MJ, Keer JT (2006) Routes to improving the reliability of low level DNA analysis using real-time PCR. BMC Biotechnol 6(1):33Google Scholar
  22. 22.
    Wilson MR, Stoneking M, Holland MM, DiZinno JA, Budowle B (1993) Guidelines for the use of mitochondrial DNA sequencing in forensic science. Crime Lab Digest 20(4):68–77Google Scholar
  23. 23.
    Goodwin C, Higgins D, Tobe SS, Austin J, Wotherspoon A, Gahan ME, McNevin D (2018) Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality. Forensic science. Med Pathol:1–6.  https://doi.org/10.1007/s12024-017-9944-8
  24. 24.
    Peccoud J, Jacob C (1996) Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys J 71(1):101–108Google Scholar
  25. 25.
    Sharpley MS, Marciniak C, Eckel-Mahan K, McManus M, Crimi M, Waymire K, Lin CS, Masubuchi S, Friend N, Koike M (2012) Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition. Cell 151(2):333–343Google Scholar
  26. 26.
    Lane N (2012) The problem with mixing mitochondria. Cell 151(2):246–248Google Scholar
  27. 27.
    Luo S-M, Schatten H, Sun Q-Y (2013) Sperm mitochondria in reproduction: good or bad and where do they go? J Genet Genomics 40(1):549–556Google Scholar
  28. 28.
    Chan DC, Schon EA (2012) Eliminating mitochondrial DNA from sperm. Dev Cell 22(3):469–470Google Scholar
  29. 29.
    Schwartz M, Vissing J (2002) Paternal inheritance of mitochondrial DNA. N Engl J Med 347(8):576–580Google Scholar
  30. 30.
    Sato M, Sato K (2013) Maternal inheritance of mitochondrial DNA by diverse mechanisms to eliminate paternal mitochondrial DNA. Biochim Biophys Acta 1833:1979–1984Google Scholar
  31. 31.
    Luo S-M, Ge Z-J, Wang Z-W, Jiang Z-Z, Wang Z-B, Ouyang Y-C, Hou Y, Schatten H, Sun Q-Y (2013) Unique insights into maternal mitochondrial inheritance in mice. PNAS 110(32):13038–13043Google Scholar
  32. 32.
    Miller FJ, Rosenfeldt FL, Zhang C, Linnane AW, Nagley P (2003) Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age. Nucleic Acids Res 31(11):e61–e61Google Scholar
  33. 33.
    Wai T, Ao A, Zhang X, Cyr D, Dufort D, Shoubridge EA (2010) The role of mitochondrial DNA copy number in mammalian fertility. Biol Reprod 83(1):52–62Google Scholar
  34. 34.
    Shin J, Kim KC, Lee DC, Lee HR, Shim JY (2017) Association between salivary mitochondrial DNA copy number and chronic fatigue according to combined symptoms in Korean adults. Korean J Fam Med 38(4):206–212Google Scholar
  35. 35.
    Alonso A, Albarrán C, Martín P, Garcıa P, Garcıa O, de la Rúa C, Alzualde A, de Simón LF, Sancho M, Piqueras JF (2003) Multiplex-PCR of short amplicons for mtDNA sequencing from ancient DNA. In: , International Congress Series. Elsevier, Amsterdam, pp 585–588Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Centre for Forensic ScienceUniversity of Technology SydneyUltimoAustralia

Personalised recommendations