Bama Yao Autonomous County, in the Guangxi Zhu- ang Autonomous Region, Guangxi province of China, is one of the world’s well-known longevity regions. Recent demographic data show that the centenarian rate is 29.9/ 100,000 in Bama County, an intriguing 27 times higher than the rate in other parts of Guangxi province and 14 times higher than that the rate of 2.19/100,000 in China in a whole. This extremely high rate of centenarians makes ‘‘the Bama longevity’’ an intriguing research topic and has attracted the attention of many scientists. There are many natural, social, environmental and genetic factors which may account for the prolonged lifespan of the Guangxi Bama population. However, to date there have not been any studies on potential molecular mechanisms behind the Bama longevity. Our objective here, then, is to attempt to explore whether and how mtDNA point mutations are associated with the Bama longevity by determining the full mtDNA sequence and by analyzing the polymorphisms of the D-loop region of Guangxi Bama population.

Genomic DNA was extracted from 200 ll whole blood samples with Blood Genome DNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manu- facturers’ manual. The extracted DNA samples were stored at -20 (cid:3)C.

PCR primers were designed using the Oligo6.0 software to cover the entire mitochondrial genome with each pair of primers spanning 2–6 kb (Table 1). The PCR reaction was performed in 50 ll solution containing 5 ll 109 LA Buffer, 2.5 mM MgCl , 0.1 mM dNTPs, 0.4 lM primers, 2 1 lg genomic DNA, and 2U LA TaqTM DNA Polymerase (Takara Biotechnology (Dalian) CO., LTD, China). Reac- tion conditions were set at 94 (cid:3)C for 5 min followed by 35

2 Primers used for No. Sequence (50?30) Start End Length the complete DNA 1 F: TTGCCCTATTACTATCCATCCTCATC 15,755 16,177 422 2 R: ACGTGTGGGCTATTTAGGCT 15,875 16,426 551 3 F: GGATACCAACAAACCTACCCA 16,342 301 528 4 R: TGTGAGCCCGTCTAAACATTTTCA 90 577 487 5 F: ACCCTAACACCAGCCTAA 438 998 560 6 F: ACACGATTAACCCAAGTCAAT 992 1,538 546 7 R: ATTTCTATCGCCTATACTTTA 1,177 1,692 515 8 F: CGTACACACCGCCCGTCAC 1,573 2,078 505 9 R: TATTGGGCTGTTAATTGTC 1,744 2,315 571 10 F: CCCAAACATATAACTGAACTCC 2,305 2,904 599 11 F: TGAAGAGGCGGGCATAACACA 2,764 3,281 517 12 R: GAGATTGTTTGGGCTACTGCT 3,152 3,584 432 13 F: AACCCTTCGCTGACGCCATAA 3,553 4,060 507 14 R: TACGGGAAGGGTATAACCAAC 3,831 4,394 564 15 F: TTCCGCTACGACCAACTCATA 4,213 4,811 598 16 F: CCGGACAATGAACCATAACCAATA 4,785 5,348 563 17 F: CATTCCTACTACTCAACT 5,230 5,769 539 18 R: GTAGAGAATAGTCAACGG 5,396 5,823 427 19 R: GTATAGTGATGCCAGCAGCTAGGA 5,872 6,419 548 20 F: TAGTTCCCCTAATAATCGGT 6,247 6,777 530 21 R: GCCGATGAATATGATAGTGAA 6,505 7,076 550 22 F: GACTGGCATTGTATTAGCA 7,070 7,649 579 23 F: CATGCAGCGCAAGTAGGTC 7,757 8,322 565 24 R: AGTCATTGTTGGGTGGTG 8,052 8,605 553 25 F: ACCTCCCTCACCAAAGCCCATA 8,581 9,131 550 26 F: GCCCTAGCCCACTTCTTACCACA 9,007 9,535 528 27 R: AAGAGTAAGACCCTCATCAA 9,303 9,909 606 28 F: GACGGCATCTACGGCTCAACATTT 9,845 10,402 557 29 R: ACTGGATAAGTGGCGTTGG 10,369 10,950 581 30 F: CCCAACAATTATATTACTACCAC 10,849 11,408 559 31 R: AGAGGATTATGATGCGACT 11,149 11,711 562 32 F: CATAGCCCTCGTAGTAACA 11,705 12,296 591 33 R: AAGGTGGATGCGACAATGG 11,866 12,323 457 34 F: AACAACATGGCTTTCTCAACT 12,323 12,923 600 35 R: AGAGTGGTGATAGCGCCTAAG 12,556 13,101 546 36 F: TCGGTTTCATCCTCGCCTTA 12,939 13,497 558 37 R: GTAAGAAGGCCTAGATA 13,412 13,869 458 38 F: CACCCTACTAAACCCCATTAAACG 13,788 14,173 385 39 F: TCACAGCCCTCGCTGTCACT 13,880 14,446 566 40 R: CCAAGGAGTGAGCCGAAGTT 14,227 14,780 553 41 F: ATTCTCGCACGGACTACAAC 14,748 15,339 591 42 F: ATTGGGACAGACCTAGTTCA 15,287 15,886 599

Taq DNA Polymerase (Applied Biosystems, American) and an Applied Biosystems 3730 automated DNA sequencer, performed by the General BioTechnology LLC (Shanghai, China). Primers for sequencing were designed to span about 500 bp and to walk from both strands of DNA helix in order to obtain the full length sequence of the

cycles of 94 (cid:3)C for 45 s, 60 (cid:3)C for 45 s and 72 (cid:3)C for 5 min. The final extension was set at 72 (cid:3)C for 10 min. PCR products were purified from 1 % low-melt agarose gel with TIANgel Midi Purification Kit (Tiangen Biotech Co., Ltd., Beijing, China). Double-stranded PCR products were sequenced directly in cycle sequencing reactions using FS

Individual Sex Age Haplogroup AC No. in 3,107 Male 110 M* :10400T HM357819 3,085 Female 110 F1:12406A 3970T 12705C 10873T HM357814 3,088 Female 108 M* :10400T HM357815 3,106 Female 107 M* :10400T HM357822 D* :5178A 10400T D4: 3010A 5178A 10400T 3,057 Female 105 M* :10400T HM357813 D* :5178A 10400T D4: 3010A 5178A 10400T 3,105 Female 104 M* :10400T HM357818 3,090 Female 103 M* :10400T HM357816 3,145 Female 103 F1: 12406A 3970T 12705C 10873T HM357820 3,104 Female 100 F1: 12406A 3970T 12705C 10873T HM357817 3,146 Female 100 M* :10400T HM357821

Table 3 Variants of

      Bama centenarian mtDNA16569bp

special PCR primers designing by Guangxi Bama full mtDNA sequence. The forward and reverse primers were 50-TTTTCGTCTGGGGGGTATGC-30 and 50-CCATCC- TACCCAGCACACAC-30, respectively, which amplified a 469 bp fragment from the mt57 to the mt525. PCR reaction content contained about 1 lg of genomic DNA, 19 PCR buffer (10 mM Tris–HCl pH 8.3, 1.5 mM MgCl , 50 mM 2 KCl), 0.2 mM dNTPs, 0.2 lM each primer, and 0.5U Taq DNA polymerase (Shanghai Sangon Biological Engineer- ing Technology & Services Co., Ltd., Shanghai, China). Reaction conditions were set at 94 (cid:3)C for 5 min followed by 35 cycles of 94 (cid:3)C for 20 s, 60 (cid:3)C for 20 s and 72 (cid:3)C for 30 s. The final extension was set at 72 (cid:3)C for 5 min. PCR products were visualized by ethidium bromide stain- ing in 1.5 % agarose gel and then sequenced by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) at fees.

PCR–RFLP