O 20 40 60 80 100 120

% mutant expected

3. If the MasterMix reagents have been stored at 4°C for longer than 2 wk or have been repetitively frozen and thawed, the PCR efficiency may be reduced. It is best to store the reagent in aliquots at -20°C.

4. The range of the standard DNA copy number chosen for the construction of the standard curve is based on the estimated copy number of the target sequences. Any measurement that falls outside of the standard DNA range should be repeated with either a higher or lower DNA concentration.


1 Anderson, S., Bankier, A. T., Barrell, B. G., et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290, 457-465.

2 Bai, R. K. and Wong, L. J. (2004) Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach. Clin. Chem. 50, 996-1001.

3 Johns, D. R. (1996) Mitochondrial DNA and disease. N. Engl. J. Med. 333, 638644.

4 Lahiri, D. and Nurnberger Jr., J. (1991) A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res. 19, 5444.

5 Liang, M. H. and Wong, L.-J. C. (1998) Yield of mtDNA mutation analysis in 2000 patients. Am. J. Med. Genet. 77, 385-400.

6 Newton, C. R., Graham, A., Heptinstall, L. E., et al. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503-2516.

7 Sambrook, J., and Russell, D.W. (2001) Molecular Cloning—Laboratory Manuals, Vol. 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

8 Shanske, S. and Wong, L.-J. C. (2004) Molecular analysis for mitochondrial DNA disorders. Mitochondrion 4, 403-415.

9 Smeitink, J., van den Heuvel, L., and DiMauro, S. (2001) The genetics and pathology of oxidative phosphorylation. Nature Rev. Genet. 2, 342-352.

10 Wallace, D. C. (1992) Disease of mitochondrial DNA. Annu. Rev. Biochem. 61, 1175-1212.

Fig. 4. (opposite page) Correlation of expected and observed percentages of A3243G mutant mtDNA from SYBR Green assay. (A) Amplification curve of the samples. (A1) Amplification curve of wild-type sequences; (A2) amplification curve of mutant sequences. The samples containing various proportions of A3243G mutation are generated by mixing the plasmid DNA containing wild-type (A3243) and mutant (A3243G) sequences. The DNA samples contain 0, 0.01, 0.1, 0.5, 1, 5, 25, 40, 45, 50, 80, 95, and 100% of mutant (A3243G) sequence, respectively, for amplification curves from left to right (A1) and from right to left (A2). (B) Correlation of observed and expected percentages of A3243G mutant sequences of the samples studied in (A). (Please see Companion CD for a color version of this figure.)

11 Wong, L.-J. C. and Lam, C. (1997) Alternative, noninvasive tissues for quantitative screening of mutant mitochondrial DNA. Clin. Chem. 43, 1241-1243.

12 Wong, L.-J. C. and Senadheera, D. (1997) Direct detection of multiple point mutations in mitochondrial DNA. Clin. Chem. 43, 1857-1861.

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