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Fig. 3 Detection of heteroplasmic A3243G mutation in mitochondrial DNA. PCR products of 643 bp (nt3116 to nt3758) containing the tRNALeu(UUR) and part of 16S rRNA and ND1 regions were analyzed with TTGE. Lanes 1 and 7: wild type; lane 2: homoplasmic T3197C polymorphism with band shift-down; lane 3: 47% heteroplasmic A3243G mutation in a homoplasmic T3197C polymorphism background; lanes 4-6: A3243G mutations with 7%, 24%, and 62% of mutant heteroplasmy.

Fig. 3 Detection of heteroplasmic A3243G mutation in mitochondrial DNA. PCR products of 643 bp (nt3116 to nt3758) containing the tRNALeu(UUR) and part of 16S rRNA and ND1 regions were analyzed with TTGE. Lanes 1 and 7: wild type; lane 2: homoplasmic T3197C polymorphism with band shift-down; lane 3: 47% heteroplasmic A3243G mutation in a homoplasmic T3197C polymorphism background; lanes 4-6: A3243G mutations with 7%, 24%, and 62% of mutant heteroplasmy.

encephalomyopathy. These studies were restricted to tRNA genes only. Recently, we have developed the temporal temperature gradient gel electrophoresis (TTGE) for the fast screening of the entire mitochondrial genome for mtDNA mutations.[26,27]

Thirty-two pairs of overlapping primers were designed to amplify the entire 16.6 kb of mitochondrial genome.1-26,27-1 The primer sequences and detailed PCR and TTGE conditions have been previously described.[26,27] The sizes of the PCR products vary from 306 to 805 bp.[26] Figure 3 shows the ability of TTGE to detect various percentages of mutant A3243G heteroplasmy. The homoplasmic T3197C polymorphism shows a band shift down (lane 2) as a result of the TA pair to CG pair change. Under optimal conditions, a single heteroplasmic mutation produces four bands representing the wild-type and mutant homoduplex bands (lower two bands in lanes 3-6) and two wild-type/mutant heteroduplex bands (upper two bands in lanes 3-6). As can be seen in this figure, the intensities of the two heteroduplex and the mutant homoduplex bands increase as the percentage of the mutant A3243G mtDNA increases from 7% to 62% (lanes 4-6 of Fig. 3). The detection of the heteroplasmic A3243G mutation in the presence of a homoplasmic T3197C background is also illustrated, where the wildtype homoduplex and all other bands are down-shifted (Fig. 3, lane 3) because of the presence of T3197C polymorphism. These results clearly demonstrate the power of TTGE to detect and distinguish homoplasmic and heteroplasmic mutations, as well as to identify het-eroplasmy in the presence of a homoplasmic polymorphism. If there is more than one heteroplasmic mutation within the same DNA fragment, TTGE will show more than four bands.[26,27]

TTGE analysis was performed on DNA specimens obtained from patients with mitochondrial respiratory disorders without identifiable common point mutations.

DNA fragments showing abnormal banding patterns were sequenced to identify the exact mutations.[26] One of the difficulties is that direct DNA sequencing sometimes cannot unequivocally detect low levels of heteroplasmic mutations. Very often, TTGE detects heteroplasmic banding patterns, but sequencing does not detect the mutations although the reduced peak height of the wild-type nucleotide may suggest the presence of mutation. In this case, a second mutation detection method, such as ASO, should be designed to confirm the putative heteroplasmic mutation.[26]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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