Abstract
We compared two different PCR strategies for the amplification of mtDNA hypervariable region 1 (HV1) with regard to the detection and interpretation of point mutation heteroplasmy in human hair roots. We monitored the level of detected heteroplasmy using direct sequence analysis. PCR amplifications were performed in duplicate on each hair root, using 62 cycles of nested PCR versus 35 cycles of direct PCR. As a previous publication reported different sensitivities of heteroplasmy detection based on the number of PCR cycles used, we were interested in whether and how different PCR amplification strategies would impact sequence quality and the detection of point heteroplasmy. We identified 12 out of 93 hair roots as heteroplasmic (7 out of 31 persons) with direct PCR, whereas 2 of these heteroplasmic events could not be identified with the nested PCR approach. Generally, the quality of the sequence electropherograms in terms of background noise was significantly lower for the nested PCR amplification strategy, leading to ambiguous results in some of the nucleotide positions. Thus, the ability to clearly distinguish a genuine mixture of two nucleotides from background noise at a heteroplasmic position was substantially greater with direct PCR amplification, which generally resulted in higher quality sequence electopherograms.
References
Lutz S, Weisser HJ, Heizmann J, Pollak S (1999) Mitochondrial heteroplasmy among maternally related individuals. Int J Legal Med 113:155–161
Calloway CD, Reynolds RL, Herrin GL Jr, Anderson WW (2000) The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age. Am J Hum Genet 66:1384–1397
Howell N, Smejkal CB (2000) Persistent heteroplasmy of a mutation in the human mtDNA control region: hypermutation as an apparent consequence of simple-repeat expansion/contraction. Am J Hum Genet 66:1589–1598
Tully LA, Parsons TJ, Steighner RJ, Holland MM, Marino MA, Prenger VL (2000) A sensitive denaturing gradient-gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region. Am J Hum Genet 67:432–443
Wilson MR, Polanskey D, Butler J, DiZinno JA, Replogle J, Budowle B (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18:662–669
Sullivan KM, Alliston-Greiner R, Archampong FIA, Piercy R, Tully G, Gill P, Lloyd-Davies C (1996) A single difference in mtDNA control region sequence observed between hair shaft and reference samples from a single donor. In: Genetic Identity Conference Proceedings of the Seventh International Symposium on Human Identification. Promega Corporation, Madison, Wisconsin, pp 126–129
Wilson MR, Polanskey D, Replogle J, Di Zinno JA, Budowle B (1997) A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes. Hum Genet 100:167–171
Holland MM, Parsons TJ (1999) Mitochondrial DNA sequence analysis—validation and use for forensic casework. Forensic Sci Rev 11:21–50
Grzybowski T (2000) Extremely high levels of human mitochondrial DNA heteroplasmy in single hair roots. Electrophoresis 21:548–553
Tully G, Lareu M (2001) Letters to the Editor: Controversies over heteroplasmy. Electrophoresis 22:180–182
Budowle B, Wallard MW, Wilson MR (2002) Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair. Forensic Sci Int 126:30–33
Parson W, Parsons TJ, Scheithauer R, Holland MM (1998) Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: application of mtDNA sequence analysis to a forensic case. Int J Legal Med 111:124–132
Szibor R, Michael L (1999) Letter to the editor: correct mitochondrial L-strand sequencing after C-stretches (W. Parson et al.). Int J Legal Med 112:348–349
Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B (1995) Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med 108:68–74
Hellmann A, Rohleder U, Schmitter H, Wittig M (2001) STR typing of human telogen hairs–a new approach. Int J Legal Med 114:269–273
Hühne J, Pfeiffer H, Waterkamp K, Brinkmann B (1999) Mitochondrial DNA in human hair shafts–existence of intra-individual differences? Int J Legal Med 112:172–175
Salas A, Lareu MV, Carracedo A (2001) Heteroplasmy in mtDNA and the weight of evidence in forensic mtDNA analysis: a case report. Int J Legal Med 114:186–190
Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Holland M, Lincoln PJ, Mayr W, Morling N, Olaisen B, Schneider PM, Tully G, Wilson M (2000) DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing. Int J Legal Med 113:193–196
Carracedo A, Baer W, Lincoln P, Mayr W, Morling N, Olaisen B, Schneider P, Budowle B, Brinkmann B, Gill P, Holland M, Tully G, Wilson M (2000) DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing. Forensic Sci Int 110:79–85
Tully G, Baer W, Brinkmann B, Carracedo A, Gill P, Morling N, Parson W, Schneider P (2001) Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles. Forensic Sci Int 124:83–91
Meyer S, Weiss G, Haeseler A von (1999) Pattern of nucleotide substitutions and rate heterogeneity in the hypervariable regions I and II of human mtDNA. Genetics 152:1103–1110
Stoneking M (2000) Hypervariable sites in the mtDNA control region are mutational hotspots. Am J Hum Genet 67:1029–1032
Grzybowski T (2001) Letters to the editor: reply. Electrophoresis 22:180–182
Acknowledgements
We thank Helga Weirich (Institute for Medical Biology and Human Genetics, University of Innsbruck) for providing the GenoM-48 Robotic Workstation, Alexandra Thaler and Harald Niederstätter (Institute of Legal Medicine, University of Innsbruck) for technical advice and valuable comments on the manuscript. We gratefully thank Barbara Rüdisser for careful review of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Brandstätter, A., Parson, W. Mitochondrial DNA heteroplasmy or artefacts—a matter of the amplification strategy?. Int J Legal Med 117, 180–184 (2003). https://doi.org/10.1007/s00414-002-0350-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00414-002-0350-7