Quantitative mitochondrial DNA mutation analysis by denaturing HPLC.
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%.


CONCLUSIONS
The quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.
Lim
Kok Seong
KS
Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093-0935, USA. kslim@ucsd.edu
Naviaux
Robert K
RK
Haas
Richard H
RH
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004272:DNA, Mitochondrial / Q000235:genetics*; D020180:Heteroduplex Analysis; D006801:Humans; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009691:Nucleic Acid Denaturation; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics; D015203:Reproducibility of Results
D004272:DNA, Mitochondrial; D012346:RNA, Transfer, Amino Acyl