A Sensitive Denaturing Gradient-Gel Electrophoresis Assay Reveals a High Frequency of Heteroplasmy in Hypervariable Region 1 of the Human mtDNA Control.

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A Sensitive Denaturing Gradient-Gel Electrophoresis Assay Reveals a High Frequency of Heteroplasmy in Hypervariable Region 1 of the Human mtDNA Control Region**Disclaimer: The opinions and assertions expressed herein are solely those of the authors and are not to be construed as official or the views of the United States Department of Defense, the United States Department of the Army, or the United States Department of Commerce. This paper is a contribution of the United States National Institute of Standards and Technology (NIST) and the United States Department of Defense and is not subject to copyright. Certain commercial equipment, instruments, or companies are identified in this paper to specify the experimental procedure. Such identification does not imply recommendation or endorsement by the government, nor does it imply that the materials or equipment identified are the best available for this purpose. Lois A. Tully, Thomas J. Parsons, Robert J. Steighner, Mitchell M. Holland, Michael A. Marino, Valerie L. Prenger The American Journal of Human Genetics Volume 67, Issue 2, Pages 432-443 (August 2000) DOI: 10.1086/302996 Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 1 DGGE gel from sensitivity experiments performed to determine the detection limit of the DGGE system. DNA from an individual with the standard reference sequence was combined with DNA from an individual with a T→C polymorphism at position 16126, prior to PCR and testing by DGGE. The concentration of the minority species could thus be intentionally varied to include 50% (lane 2), 10% (lane 3), 5% (lane 4), 2% (lane 5), 1.3% (lane 6), and 1% (lane 7). Lanes 1 and 8 represent single PCR products from the reference sequence and the 16126C polymorphism, respectively. Although the homoduplex band representing the reference sequence was not visible at a level ≤10%, the heteroduplexes were clearly seen at levels as low as 1%. (Reprinted with permission from the American Society for Testing and Materials.) The American Journal of Human Genetics 2000 67, 432-443DOI: (10.1086/302996) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 2 Least-squares line fit for known and calculated heteroplasmic proportions. A series of simulated heteroplasmic samples with various proportions of the minor species were analyzed by DGGE. The known heteroplasmic proportions were plotted against the calculated proportions after densitometric analysis of DGGE gel photographs (coefficient of correlation .9026). The American Journal of Human Genetics 2000 67, 432-443DOI: (10.1086/302996) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 3 mtDNA heteroplasmy at position 16093. The original PCR product (top) appears to be heteroplasmic (C/T) at position 16093. The sequence data from both homoduplexes (middle and bottom) confirm the heteroplasmic position and sequence. The American Journal of Human Genetics 2000 67, 432-443DOI: (10.1086/302996) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 4 Homoduplex and heteroduplex DNA sequences. Sequence electropherograms of reamplified homoduplex (top) and heteroduplex (bottom) DNA from a simulated heteroplasmic sample (prepared by combining two homoplasmic samples differing at a single position) are shown. Comparison of the sequence data allows for identification of the heteroplasmic position. The American Journal of Human Genetics 2000 67, 432-443DOI: (10.1086/302996) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 5 Potential heteroplasmy at position 16192. The sequence electropherograms of reamplified homoduplex (top) and heteroduplex (bottom) DNA are shown. A very low level of a second sequence (C) is seen at position 16192 in the heteroduplex. However, this is insufficient for identification of heteroplasmy. The American Journal of Human Genetics 2000 67, 432-443DOI: (10.1086/302996) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 6 Sequence electropherograms after analysis of heteroduplex DNA by second-round DGGE. The sequences of the lower band (top) and upper band (bottom) confirmed 16192 as the heteroplasmic position. The American Journal of Human Genetics 2000 67, 432-443DOI: (10.1086/302996) Copyright © 2000 The American Society of Human Genetics Terms and Conditions