The PCR amplification was carried out using the following program: 95°C for 4 minutes - [94°C for 30 seconds - 60°C (-0.5°C/cycle) for 50 seconds - 72°C for 90 seconds] *11 cycles [94°C for 30 seconds - 55°C for 50 seconds - 72°C for 90 seconds]*24 cycles - 72°C for 10 minutes - 4°C. The final concentrations of each forward and reverse primer for mtDNA SNPs are as follows: For Multiplex 1, 12705 and 12811 are 0.33 mM; 12406 and 12358 are 0.35 mM; 7600 and 10400 are 0.53 mM; 8281 is 0.6 mM; 14569 is 0.67 mM; 9296 is 0.7 mM. For Multiplex 2, 4491, 8020, 8584, and 8684 are 0.2 mM; 5417 and 5465 are 0.25 mM; 1107 is 0.27 mM; 3010, 3970, 4833, 5178, 7196, 9824, 10646, 10873, and 15662 are 0.3 mM; 15235 is 0.37 mM; 3705, 8964, 13263, and 13590 are 0.4 mM; and 14979 is 0.47 mM; while1736 is 0.5 mM.

After the PCR amplification, the amplification products from both multiplexes were mixed and incubated with Exonuclease I (New England Biolabs, Ipswich, MA) and shrimp alkaline phosphatase (SAP) (Promega, Madison, WI) for eighty minutes at37°C followed by fifteen minutes at75°C for enzyme inactivation. Then multiplex primer extension reactions were carried out by SNaPshot Multiplex Ready reaction mix (Applied Biosystems, Foster City, CA), Sequencing Buffer5X (Applied Biosystems), and primers extension mix with the multiplex primer extension cycling conditions:96°C for one minute - [96°C for ten seconds -52°C for five seconds-60°C for thirty seconds] *28 cycles -4°C; the final concentrations of each extension primer are also given:1107,1736,3010,3705,3970,4491,4833,5178,7196,7600,8020,8584,8684,8964,9824,10646,10873,12811,13263,13590,15235, and 15662 are 0.16 mM; 5417 is 0.33 mM; 12406 is 0.37 mM; 5465, 12358, 12705, and 14979 are 0.41 mM; 14569 is 0.45 mM; and 8281, 9296, and 10400 are 0.49 mM. After the SNaPshot reaction, the excess ddNTPs were inactivated by treating the reaction with shrimp alkaline phosphatase (Promega) for one unit followed by electrophoresis of final minisequencing products on the ABI3130xl Genetic Analyzer (Applied Biosystems). Genemapper ver.4.0 software was used to analyze the data.

Discussion: In this study, we conducted a population-based case-control study to explore the effects of mtDNA haplogroups on the phenomenon of extremely long life in a Chinese population. Crucial to our approach is theThe text appears to be a table and its description. There are no spelling or grammatical errors to correct. However, some of the words are split across two lines, and there are unnecessary hyphens that need to be removed. Here's the processed text: 

0.779 1.000 0.949 G2 13 (2.8%) 18 (1.9%) 7 (1.5%) 0.304 0.175 0.153 M7 35 (7.6%) 67 (7.2%) 36 (7.8%) 0.827 0.902 0.955 M7b 18 (3.9%) 35 (3.8%) 22 (4.8%) 0.921 0.518 0.587 M8 47 (10.2%) 112 (12.1%) 42 (9.1%) 0.283 0.577 0.847 M8a 10 (2.2%) 37 (4.0%) 12 (2.6%) 0.074 0.666 0.457 C 22 (4.8%) 43 (4.6%) 17 (3.7%) 0.928 0.413 0.485 M9 1 (0.2%) 20 (2.2%) 8 (1.7%) 0.005 0.019 0.027 M10 9 (1.9%) 15 (1.6%) 8 (1.7%) 0.662 0.807 0.753 N9 
22(4 .8% )67(7 .2% )40(8 .6% )   .075   .018   .018 N9a 
18(3 .9% )51(5 .5% )31(6 .7% )   .190   .056   .058 A 
35(7 .6% )66(7 .1% )26(5 .6% )   .770   .233   .288 F 
77(16.6%) 138 (14.9%) 61 (13.2%) 0.401 0.140 0.147 F1 45 (9.7%) 81 (8.7%) 36 (7.8%) 0.552 0.295 0.304 B 
77 (16.8%) 152 (16.5%) 91 (19.7%) 0.898 0.251 0.311 B5 
29 (6.3%) 39 (4.2%) 20 (4.3%) 0.097 0.186 0.117 B5a 
16 (3.5%) 22 (2.4%) 8 (1.7%) 0.245 0.098 0.086 B5b 
13 (2 .8% )16(1 .7% )12(2 .6% )   .186   .839   .357 B4a 
9 (1 .9% )31(3 .4% )17(3 .7% )   .139   .112   .105 B4b 
8(1 .7% )24(2 .6% )11(2 .4% )   .310   .487   .430 

It has never been reported elsewhere that B4a is negatively correlated with ages in Rugao population. The correlation between mtDNA haplogroups and longevity is consistent with the hypothesis that mtDNA variations have a higher impact on male than on female longevity [37]. This gender difference could be one of the consequences of the maternal inheritance of mtDNA, which in turn implies a higher relaxation and less purging of male-specific mitochondrial phenotype, being males evolutionary dead ends for mtDNA. Interestingly, the haplogroup D4 was not enriched in male Rugao subjects, but only in female subjects. Coincidentally, longevity also shows maternal inheritance [38], and the female-to-male ratio is 3.5:1 in Rugao long-lived subjects. The fundamental mechanism of this phenomenon and the female correlation warrants further exploring.

The population was relatively homogeneous related to environmental exposures, which decreased the selection biases for case-control matching. Finally, although our study included 463 cases and two control groups, interpreting the results is also limited by the relatively insufficient sample size. For example, our sample size had the power of 0.91 and 0.65 to detect the differences between longevity subjects and elderly subjects, between longevity subjects and middle-aged subjects, respectively, given the significant level of 0.05. However, when applying a conservative Bonferroni correction for multiple testing, theCity, Jiangsu Province, Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM et al. (1990) Rapid simple method for purification of nucleic acids. J Clin Microbiol 28(3): 495-503.

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