Measurement of Mutant Heteroplasmy

The standard curves for both wild-type and mutant mtDNA are always included in each run. The copy number of the target sequence in the sample is calculated from the threshold cycle number and the standard curve. The proportion of mutant A3243G sequence is calculated from the copy number of the wild-type and mutant sequences. Alternatively, the proportion of the mutant mtDNA can be calculated from ACT (CTnormal-CTmut) using the formula: proportion of mutant = 1/ (1 + 1/2ACT). The observed and expected percentage heteroplasmy for both TaqMan real-time qPCR and SYBR Green ARMS realtime qPCR should be in good agreement (Figs. 3 and 4). However, for the very low or very high heteroplasmy, the ARMS qPCR is more sensitive than TaqMan qPCR.

Fig. 3. Correlation of expected and observed percentages of A3243G mutant mitochondrial DNA from TaqMan® assay. The samples were mixtures of plasmid DNA samples with known percentage of mutant load. (Please see Companion CD for a color version of this figure.)

Fig. 3. Correlation of expected and observed percentages of A3243G mutant mitochondrial DNA from TaqMan® assay. The samples were mixtures of plasmid DNA samples with known percentage of mutant load. (Please see Companion CD for a color version of this figure.)

1. In the TaqMan assay, the wild-type and mutant probes differ by only one nucle-otide, nonspecific binding of the wild-type probe to mutant target sequence or vice versa makes the determination of low percentage heteroplasmy less accurate. The ARMS qPCR does not have this problem. The advantage of TaqMan assay is that any primer dimer will not be detected. This could be a disadvantage in the real-time ARMS qPCR SYBR Green assay, because the detection is based on the intercalation of SYBR Green dye to any double-stranded DNA products, including primer-dimers. However, in the absence of primer-dimer, ARMS primer specifically amplifies the target specific sequence and is sensitive in detecting low percentage of heteroplasmy.

2. MasterMix reagents from different manufacturers may have different amplification efficiencies and, thus, CT values. The concentrations of the primers and the DNA template used in the real-time PCR reaction may also affect the results. Therefore, in order to compare data from different runs, standard curves and control specimens that have been analyzed before should always be included in each run. In our experience, for TaqMan probe assay, Invitrogen's Platinum qPCR SuperMix-UDG is more efficient in the amplification of DNA than TaqMan Universal PCR Master Mix from Applied Biosystems Inc. under the same PCR conditions. However, the SuperMix-UDG is less accurate than Universal MasterMix in the detection of low percentages of heteroplasmies. For ARMS SYBR Green assay, Invitrogen's Platinum SYBR Green qPCR SuperMix-UDG is also more efficient than TaqMan Universal PCR Master Mix in DNA amplification under the same PCR conditions.

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