PCR amplifications were carried out in a final reaction volume of 20 The FL primer was a 38-mer oligonucleotide, consisting of an 18-base sequence of an universal forward sequencing primer (-21M13, 5'-TGTAAACGCGGCCAGT-3') connected on its 5' side to the 3' side of a 20-base L-strand-specific sequence The sequence of primers H and FL are shown in The first PCR DNA templates for the sequence analysis of the entire mitochondrial genome were amplified as 60 overlapping segments (1 to 60), each of approximately 600-1000 bp, by a symmetric PCR method with the primer pairs (FL and H primers) shown in The amplified fragments were analyzed by electrophoresis on a 1% agarose gel and visualized by staining with ethidium bromide. Table 2 shows the primer concentration and annealing temperature used for amplification of each fragment. The PCR conditions used were the following: an initial denaturation step at 94✬ for 5 min, followed by 40 cycles of denaturation at 94✬ for 15 s, annealing at 52-62✬ for 15 s, and extension at 72✬ for 3 min, with a final extension of 10 min at 72✬. ΜM concentration of each primer, and 1 unit of Taq DNA polymerase (Takara, Shiga, Japan). MgCl 2, 0.2 mM concentration of each dNTP, 0.5 or 1.0 Μl, containing 200 ng human genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM PCR amplification was carried out in a final reaction volume of 40 The sequences of primers L and H are shown in The oligonucleotide primers, synthesized and purified by gel filtration, were obtained from Bio-Synthesis (Lewisville, Texas). The entire mitochondrial genome was amplified as 6 fragments (A to F), each approximately 3.0 kb in length, by a symmetric PCR method with the primer pairs (L and H primers) shown in The entire mitochondrial genome was amplified as 6 fragments by the first PCR, and then 60 overlapping segments were amplified by the second PCR, essentially as described previously (Tanaka et al., Methods in Enzymology, 1996). ![]() Venous blood (2 ml) was collected from each subject into a tube containing 50 mM EDTA and genomic DNA was isolated from the total blood samples by the standard phenol-chloroform method, or by use of a DNA isolation kit Dr GenTLE (Takara, Osaka Japan) or an automated DNA extraction system MagExtractor MFX-2000 (Toyobo,
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