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Clinical Chemistry 53: 1046-1052, 2007. First published April 19, 2007; 10.1373/clinchem.2006.083303
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(Clinical Chemistry. 2007;53:1046-1052.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC

Kok Seong Lim1,a, Robert K. Naviaux2,3 and Richard H. Haas1,3

Departments of1 Neurosciences, 2 Medicine, and 3 Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA.

aAddress correspondence to this author at: University of California San Diego School of Medicine, 9500 Gilman Dr. B112, La Jolla, CA 92093-0935. Fax 619-543-7868; e-mail kslim{at}ucsd.edu.

Background: In recent years, denaturing HPLC (DHPLC) has been widely used to screen the whole mitochondrial genome or specific regions of the genome for DNA mutations. The quantification and mathematical modeling of DHPLC results is, however, underexplored.

Methods: We generated site-directed mutants containing some common mutations in the mitochondrial DNA (mtDNA) tRNA(leu) region with different mutation loads and used PCR to amplify the gene segment of interest in these mutants. We then performed restriction digestion followed by slow reannealing to induce heteroduplex formation and analyzed the samples by use of DHPLC.

Results: We observed a quadratic relationship between the heteroduplex peak areas and mutant loads, consistent with the kinetics of heteroduplex formation reported by others. This was modeled mathematically and used to quantify mtDNA mutation load. The method was able to detect a mutation present in a concentration as low as 1% and gave reproducible measurements of the mutations in the range of 2.5%–97.5%.

Conclusion: The quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.




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K. S. Lim, R. K. Naviaux, S. Wong, and R. H. Haas
Pitfalls in the Denaturing High-Performance Liquid Chromatography Analysis of Mitochondrial DNA Mutation
J. Mol. Diagn., January 1, 2008; 10(1): 102 - 108.
[Abstract] [Full Text] [PDF]


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