Introduction process (Papa and Skulachev, +;;,), Brierley et al., +;;,, ErLee and Wei, +;;,, Miquel, +;;:, Brand, Kowald and Kirkwood, (((), Meloy, (((), Rustin et al., (((>). Oxygen free radicals, usually referred to as reactive oxygen species are the major source of oxidative damage within the cell and lie at the heart of one of the most prominent and plausible theories postulated, the free radical theory of ageing (Beckman and Ames, +;;:, Cadenas and Davies, (((), Owen; Sastre et al., 7(((>). The main source of FROS within the cell is the aerobic metabolic pathway located within the mitochondrion. The ROS produced cause cumulative damage to cellular constituents - DNA, RNA, proteins and lipids - that is believed to result in ageing and eventual death. The mitochondrial DNA genome (mtDNA) is a closed circular double-stranded genome of +3%3; bp that encodes for tRNAs, polypeptides and is estimated to undergo mutations at least 3 times faster than nuclear DNA (aDNA). There are at least two factors implicated in promoting this high rate of mutation: (1) there are no histone proteins to protect mtDNA and (2) there is only a very inefficient repair mechanism against damaged DNA. DNA is located in an area termed the control region. The control region is the variable region of the human mtDNA genome and is the location of "the loop" (D-loop). The number of features exhibited by mtDNA (polymorphic maternal mode of inheritance, lack of recombination, and relatively small genome) led to its analysis by molecular approaches being widely exploited in anthropological studies, providing clearer insight into human evolution, origins, and patterns of migration (Torroni et al., +;;).Pengman et al. have established that numerous mtDNA variants/haplotypes, defined by a restriction enzyme that exists within the European Caucasian population, can be differentiated into nine mtDNA haplogroups: I, E, D, G, J, and others, based on polymorphisms associated with longevity in humans. De Benedictis and colleagues reported a statistically significant increase in the frequency of the broad haplogroup, including subgroups I, J, and T, among male centenarians from the Avon North region of Italy compared to younger males from the same region. Two other independent studies identified increases in the frequencies of mitochondrial polymorphisms at position 12395 in Japanese centenarians and in French centenarians. Successful aging was assessed in this study. For analysis, a 344 bp section of the mtDNA was targeted and subjected to high-density restriction enzyme analysis as outlined previously (Torroni et al., 1993). This region of mtDNA was selected for a number of reasons. Firstly, it contained part of our cohort of control and aged cases and permitted the construction of a table of genetic haplotypes incorporating various polymorphic restriction sites encountered when bands deviated from the predicted patterns inferred from the Anderson sequence (Anderson et al., 1981). Secondly, it contains the polymorphic restriction site which defines the European haplogroup and would therefore allow us to determine whether this haplogroup is also increased in our aged Irish population. Thirdly, the absence of recombination with respect to the mtDNA infers that any other ancestral polymorphism outside of the region analyzed and possibly involved in aging would be tightly linked to one or more of the various haplotypes/groups created for this region and contribute indirectly to the overall analysis. In addition to this analysis, the frequency of mitochondrial nucleotide polymorphisms at positions 12395 and 10398 were examined in our control and aged populations in an attempt to verify the previous findings of significant associations and to establish whether these polymorphisms are also significantly increased in the aged community. Subjects were chosen at random from the DNA bank of normal healthy Caucasian individuals from the Northern Ireland population stored at the Northern Ireland Histocompatibility and Immunogenetics Laboratory of Belfast City Hospital. The study involved 47 aged consecutive samples (23 female, 24 male, with an age-range of 70-98 years old) from the Belfast Elderly Longitudinal Free-living Ageing Study. All samples were healthy Caucasian individuals from the Northern Ireland population displaying no signs of age-related disorders, still living independently and approximately 77 years old on average (Ligthart et al., 2014). In the case of the polymorphism study, DNA samples were derived from a cohort of unrelated Asian individuals (Singapore Chinese, age-range 20-80 years old) accessed from the DNA archival bank at NUS and also used to provide positive and negative controls for this particular polymorphism and therefore verify our sequence-specific probe results. PCR amplification of mtDNA region spanning nucleotides 4378-403 was essentially those described by Torroni et al. but extended to improve the specificity and sensitivity of the selected region of the mtDNA.Psincluded 1; the BONE, JOD entire Sytb y and ay NS ND6 genes, Karen Small Ape and a section of the NDS oegene were used to identify the exact Ce ONt nucleotide variation causing the presence or absence of polymorphic restriction sites. Amplification primers for the mtDNA region D-loop were used to sequence the genome according to the published sequence from Anderson et al. Cing primers were used for polymorphisms encountered in this study. Successful amplification was confirmed using gel electrophoresis on a Neagarose gel stained with methidium bromide. After successful amplification, each product was subjected to Hinfl, Hpal, Mbol, Rsal, BamHI, and Haell restriction enzymes. Purified products were sequenced using appropriate sequencing primers shown in Table. Statistical analysis was performed using Fisher's exact probability tests to assess frequency differences between aged and control groups for haplotypes, groups of related haplotypes, and