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Factors affecting the detection and quantification of mitochondrial point heteroplasmy using Sanger sequencing and SNaPshot minisequencing

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Abstract

Mitochondrial DNA analysis plays an important role in forensic science as well as in the diagnosis of mitochondrial diseases. The occurrence of two different nucleotides at the same sequence position can be caused either by heteroplasmy or by a mix of samples. The detection of superimposed positions in forensic samples and their quantification can provide additional information and might also be useful to identify a mixed sample. Therefore, the detection and visualization of heteroplasmy has to be robust and sensitive at the same time to allow for reliable interpretation of results and to avoid a loss of information. In this study, different factors influencing the analysis of mitochondrial heteroplasmy (DNA polymerases, PCR and sequencing primers, nucleotide incorporation, and sequence context) were examined. BigDye Sanger sequencing and the SNaPshot minisequencing were compared as to the accuracy of detection using artificially created mitochondrial DNA mixtures. Both sequencing strategies showed to be robust, and the parameters tested showed to have a variable impact on the display of nucleotide ratios. However, experiments revealed a high correlation between the expected and the measured nucleotide ratios in cell mixtures. Compared to the SNaPshot minisequencing, Sanger sequencing proved to be the more robust and reliable method for quantification of nucleotide ratios but showed a lower detection sensitivity of minor cytosine components.

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Acknowledgments

This study was funded by the state of Baden-Württemberg (Competence Centre “Legal Medicine”).

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Authors and Affiliations

  1. Institute of Legal Medicine, Freiburg University Medical Center, Albertstr. 9, 79104, Freiburg, Germany

    Jana Naue, Timo Sänger, Ulrike Schmidt & Sabine Lutz-Bonengel

  2. Institute of Legal Medicine, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany

    Rachel Klein

Authors
  1. Jana Naue
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  2. Timo Sänger
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  3. Ulrike Schmidt
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  4. Rachel Klein
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  5. Sabine Lutz-Bonengel
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Corresponding author

Correspondence to Jana Naue.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1

Primers for PCR, Sanger sequencing, and SNaPshot minisequencing used in this study. (PDF 14 kb)

Online Resource 2

Nucleotide incorporation and display of heteroplasmy at position 16093. (PDF 1.78 mb)

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Naue, J., Sänger, T., Schmidt, U. et al. Factors affecting the detection and quantification of mitochondrial point heteroplasmy using Sanger sequencing and SNaPshot minisequencing. Int J Legal Med 125, 427–436 (2011). https://doi.org/10.1007/s00414-011-0549-6

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  • DOI: https://doi.org/10.1007/s00414-011-0549-6

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