Received on November 17, 2006
Accepted on March 27, 2007

Molecular Diagnostics and Genetics

Quantitative DNA Mutation Analysis by Denaturing HPLC

¹ Department of Neurosciences, University of California San Diego, La Jolla, CA
² Department of Medicine, and Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA
³ Department of Neurosciences, and Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA

^* To whom correspondence should be addressed. 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.