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TECHNICAL ANNEX

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1. Isolation of Genomic DNA
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, Shiga, Japan). 
2. Amplification of DNA
  • 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).
  • First PCR
    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 Table 1. The oligonucleotide primers, synthesized and purified by gel filtration, were obtained from Bio-Synthesis (Lewisville, Texas). The sequences of primers L and H are shown in Tables 3 and 4, respectively. PCR amplification was carried out in a final reaction volume of 40 µl, containing 200 ng human genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM concentration of each dNTP, 0.5 or 1.0 µM concentration of each primer, and 1 unit of Taq DNA polymerase (Takara, Shiga, Japan). The PCR conditions used were the following: an initial denaturation step at 94ºC for 5 min, followed by 40 cycles of denaturation at 94ºC for 15 s, annealing at 52-62ºC for 15 s, and extension at 72ºC for 3 min, with a final extension of 10 min at 72ºC. Table 2 shows the primer concentration and annealing temperature used for amplification of each fragment. The amplified fragments were analyzed by electrophoresis on a 1% agarose gel and visualized by staining with ethidium bromide.
  • Second PCR
    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 Table 1. The sequence of primers H and FL are shown in Tables 4 and 5, respectively. 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 (Table 5). PCR amplifications were carried out in a final reaction volume of 20 µl, containing 200 ng of the first PCR product, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM concentration of each dNTP, 1.0 µM concentration of each primer, and 0.5 units of Taq DNA polymerase (Takara, Shiga, Japan). The PCR conditions used were the same as for the first PCR except that the annealing temperature used was 60ºC. These second PCR products were purified by use of MultiScreen-PCR Plates (Millipore, Bedford, MA), and then the qualities of DNA templates were examined by electrophoresis on a 1.2% agarose gel after staining with ethidium bromide by use of a Ready-To-Run Separation Unit (Amersham Pharmacia Biotech AB, San Francisco, CA).

3. Sequence analysis and identification of mtSNP
Sequence reactions were performed by use of the second PCR template, -21M13 forward primer, and a BigDye Terminator Cycle Sequence Ready Reaction Kit version 1.0, 3.0, or 3.1 (Applied Biosystems, Foster City, CA). The following PCR conditions were used: an initial denaturation step at 96ºC for 5 min, followed by 25 cycles of denaturation at 96ºC for 10 s, annealing at 50ºC for 5 s, and extension at 62ºC for 4 min. After the sequence reaction, excess dye terminators were removed by gel filtration on a MultiScreen-PCR HV Plate (Millipore). The purified DNA samples were dried and suspended in the template suppression reagent (TSR) or formamide from Applied Biosystems. The dissolved DNA samples were heated at 95ºC for 2 min for denaturation, and then immediately cooled on ice. Sequences were analyzed with an automated DNA sequencer Prism 310 or 377 from Applied Biosystems. Sequence analysis was performed by use of Sequencing Analysis Program version 4.1 software (Applied Biosystems). Complete sequences were aligned, assembled, and compared with the program Sequencher 4.1 (Gene Codes, Ann Arbor, MI). For verification, visual inspection of each candidate mtSNP was carried out. At least 2 overlapping DNA templates amplified with different primer pairs were used for identification of each mtSNP.
4. Reverse sequencing of 5 segments 
In some cases, the light-strand sequences could not be determined on the 3' side of long stretches of C due to T>C transitions at positions 310, 961, and 16189 or due to the poly-C sequences at 568-573 and 5895-5899. For such cases, the heavy-strand (reverse) sequences were determined for 5 segments (02, 03, 21, 58, and 60). For this purpose, the primers H81, H528, H807, and H6158 listed in Table 6 were used for sequencing the second PCR products with a BigDye terminator cycle sequence ready reaction kit version 3.1 from Applied Biosystems. 
The segment 03 was amplified from the first PCR product as the template by use of the FL700 and the T7H1162 primers. The T7H1162 primer was a 40-mer oligonucleotide, which was a 20-base sequence of a T7 promoter (5'-TAATACGACTCACTATAGGG-3') connected on its 5' side to the 3' side of a 20-base H-strand-specific sequence (5'-CACCGCCAGGTCCTTTGAGT-3'). The sequence reaction was performed at 37C for 1 hr by use of the second PCR product (FL700-T7H1162) as the template with a CUGA7 sequencing kit (Nippon Genetech, Tokyo, Japan) designed for the ABI Prism 310 genetic analyzer. After removal of excess dye terminators by gel filtration on a Centri-Sep spin column (Applied Biosystems), the sample was applied to the ABI Prism 310 genetic analyzer.
5. Comparison with the revised Cambridge reference sequence (rCRS):
Each of the mtDNA sequences was compared with the original Cambridge sequence (Anderson et al., Nature, 1981) and the revised Cambridge reference sequence (rCRS) (Andrews et al., Nat Genet, 1999). 
6. Subject groups
  • Centenarian group (TC and GC): This group consists of 96 centenarians over 100 years of age who were registered by the Department of Gerontology, Keio University School of Medicine, in Tokyo (TC group: n=85, 60 females and 25 males) or by Gifu International Institute of Biotechnology (GC group: n=11, 6 females and 5 males). 
  • Parkinson's disease group (PD): The PD group consists of 96 patients with Parkinson's disease (mean age of 62.1 ± 8.9 years; range, 39-81 years) who were diagnosed at the Department of Neurology, Juntendo University School of Medicine, in Tokyo (53 females and 43 males).
  • Alzheimer's disease group (KA): The KA group comprises 96 patients with Alzheimer's disease (mean age of 76.5 ± 9.7 years; range, 47-93) who were diagnosed at the Department of Neurology, Nihon Medical School, in Chiba (n=96, 76 females, and 20 males).
  • Obesity group (ON): The ON group consists of 96 young obese male subjects (mean age of 21±2 years; range, 18-25 years) with a body mass index (BMI) of 30.1±2.5, who were registered at the Research Center of Health, Physical Fitness and Sports, Nagoya University, in Nagoya.
  • Healthy non-obese group (HN): The HN group consists of 96 young healthy non-obese male subjects (mean age of 20±3 years; range, 18-25) with a body mass index (BMI) of 20.2±2.3, who were registered at the same location as the obese group.
  • Diabetes group (ND) in Nagoya: The ND group consists of 96 patients with type-2 diabetes mellitus (mean age of 58±5 years; range, 43-65), who were registered at the same location as the obese group (n=96, 42 females and 54 males).
  • Data from Ingman et al. (Ingm): The complete mtDNA sequences of 53 humans of diverse origins were reported by Ingman et al. (Ingman M, Kaessmann H, Paabo S, Gyllensten U, Mitochondrial genome variation and the origin of modern humans. Nature 408: 708-713, 2000). For further information, please visit: http://www.genpat.uu.se/mtDB/, http://www.genpat.uu.se/anthropology/ and http://www.actionbioscience.org/evolution/ingman.html.
  • Diabetes group (JD) in Tokyo: The TD group comprises 96 type-2 diabetes patients with severe vascular involvement (mean age of 65±10 years; range, 43-92), who were registered at the Department of Internal Medicine, Juntendo University School of Medicine, in Tokyo (n=96, 48 females and 48 males).
7. Contributors
  • Masashi Tanaka (GiiB)
  • Noriyuki Fuku (GiiB, JST)
  • Raita Hirose (GiiB, JST)
  • Takeshi Takeyasu (GiiB, Nagoya University)
  • Li-Jun Guo (GiiB, Nagoya University)
  • Osamu Ogawa (Juntendo University)
  • Miyuki Kurata (GiiB)
  • Yoshiji Yamada (GiiB)
  • Masayo Shamoto (GiiB, JST, NILS)
  • Harm-Jan W. Borgeld (GiiB, Institute of Applied Biochemistry)
  • Kunio Yagi (GiiB, Institute of Applied Biochemistry)
8. Collaborators
  • Yoshiharu Oshida (Nagoya University)
  • Yuzo Sato (Nagoya University)
  • Yasumichi Arai (Keio University)
  • Nobuyoshi Hirose (Keio University)
  • Nobutaka Hattori (Juntendo University)
  • Yoshikuni Mizuno (Juntendo University)
  • Shigeo Ohta (Nihon Medical School)
  • Takeshi Nomiyama (Juntendo University)
  • Hajime Matsunaga (Juntendo University)
  • Yasushi Tanaka (Juntendo University)
  • Ryuzo Kawamori (Juntendo University)
  • Wakako Maruyama (NILS)
  • Hiroshi Shimokata (NILS)
  • Jian-Sheng Gong (GiiB, NILS)
  • Jin Zhang (GiiB, Caltech)
9.Advisors
  • Yasuo Kagawa (Kagawa Nutrition University, Jichi Medical School)
  • Nobuo Tanaka (Tokyo Institute of Technology)
  • Kazuo Umetsu (Yamagata University)
  • Yuichi Goto (National Center of Neurology and Psychiatry)
  • Naruya Saito (National Institute of Genetics)

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