Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct:Chapter 19:19.8.1-19.8.12.
doi: 10.1002/0471142905.hg1908s71.

Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy

Taosheng Huang  1
Affiliations

Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy

Taosheng Huang. Curr Protoc Hum Genet. 2011 Oct.

Abstract

This protocol describes the methodology to characterize mitochondrial DNA (mtDNA) heteroplasmy by parallel sequencing. Mitochondria play an important role in essential cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondria genomes. Mutant and wild-type mtDNA may co-exist as heteroplasmy, and cause human disease. The purpose of this protocol is to simultaneously determine mtDNA sequence and quantify the heteroplasmic level. This protocol includes a two-fragment mitochondrial genome DNA PCR amplification. The PCR product is then mixed at an equimolar ratio. The samples are then barcoded and sequenced with high-throughput, next-generation sequencing technology. This technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the level of heteroplasmy.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Serial deletion of genomic DNA to test PCR sensitivity
Lane 1, 10 ng; lane 2, 2 ng; lane 3, 400 pg; lane 4, 80 pg; lane 5, 16 pg; lane 6, H20. Note that clear PCR results were obtained at the 16 pg dilution.
Fig. 2
Fig. 2. Coverage maps for 5% mixture sample in Test 1 (top) and Test 2 (bottom)
The X-axis represents the position of nucleotide on the mitochondrial genome (rCRS as reference genome, 16.568 kb), and the Y-axis stands for the fold of coverage for each nucleotide position.
See this image and copyright information in PMC

References

    1. Andrews RM, Kubacka I, et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet. 1999;23(2):147. - PubMed
    1. Bai RK, Wong LJ. Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach. Clin Chem. 2004;50(6):996–1001. - PubMed
    1. Bannwarth S, Procaccio V, et al. Surveyor Nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects. Hum Mutat. 2005;25(6):575–82. - PubMed
    1. Bentley DR. Whole-genome re-sequencing. Curr Opin Genet Dev. 2006;16(6):545–52. - PubMed
    1. Coskun PE, Beal MF, et al. Alzheimer’s brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication. Proc Natl Acad Sci U S A. 2004;101(29):10726–31. - PMC - PubMed

Publication types

Substances

LinkOut - more resources