Bp mtDNA Research Articles

A Simplified Method for Mitochondrial DNA Extraction from Head Hair Shafts

DNA isolation from hair shafts can involve a number of steps, each of which adds time to the procedure and increases the risk of contamination. A simple alkaline digestion procedure that directly dissolves hairs was developed and compared with a widely used glass grinding/organic extraction method, using samples collected from 30 volunteers with varying population ancestries, hair colors, and hair treatments. A 203 bp mtDNA product could be amplified from 90% of samples extracted by alkaline digestion and 73% of hairs extracted by glass grinding. DNA obtained from alkaline digested hair generated equal or greater amplification success for virtually all criteria examined, and mtDNA sequences matched buccal control sequences in all cases. The two methods were similar in DNA yield (amplification success at template dilution) and quality of DNA obtained (amplicon length). Alkaline digestion of hair shafts required 6-7 h to complete, compared to 22-24 h for glass grinding, and proved a less laborious yet equally robust method for mtDNA extraction.

Taenia saginata: Differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated.. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.

Dispersal and phylogeography of the agamid lizardAmphibolurus nobbiin fragmented and continuous habitat

Approximately 90% of native vegetation has been cleared for agriculture in central New South Wales, Australia. Habitat loss has reduced and fragmented populations of the agamid lizard Amphibolurus nobbi. We compared genetic structure of populations of this species in an unmodified landscape with those from small nature reserves and linear remnants in farming areas. We ask: Is there evidence for reduced dispersal and population fragmentation among farm populations? Using 2008 bp mtDNA sequences and allozyme electrophoresis, we found that small populations in farming areas had as much genetic variation as populations in nature reserves. Application of nested clade analysis (NCA) indicated isolation-by-distance effects among populations from uncleared areas, but not among populations within farming locations. The genetic evidence therefore implied a high level of migration in the cleared landscapes. High dispersal after fragmentation may have resulted from either a burst of movement at the time of land clearing with dragons from many sources finding refuge in a few remnants, or from ongoing rapid dispersal through unsuitable habitat. A phylogeny based on mtDNA revealed that A. nobbi populations in the study area are deeply divided into two reciprocally monophyletic groups. Although we did not sample the entire species range, one of these evolutionarily significant units was only detected in remnant vegetation in the agricultural landscape. Therefore, a substantial subclade of this species may be vulnerable to extinction. Our findings emphasize that local populations of widespread species can harbour important intraspecific genetic diversity, supporting the case for maintaining widespread species throughout production landscapes.

The Use of a 3895 bp Mitochondrial DNA Deletion as a Marker for Sunlight Exposure in Human Skin

Previous work has examined the use of mitochondrial DNA (mtDNA) damage as a biomarker of cumulative sun exposure in human skin. These studies have simply compared mtDNA damage between sun-protected and sun exposed skin. This approach is limited because non-melanoma skin cancer (NMSC) is predominantly formed on body sites which are 'usually' sun exposed as opposed to sites which are 'occasionally' sun exposed and as such they differ in their cumulative ultraviolet (UV) exposure. This study addresses this limitation by investigating the frequency of occurrence of a rarely reported 3895 bp mtDNA deletion in 104 age-matched human skin samples taken from different sun-exposed body sites. There was a significant increase in the deletion frequency with increasing UV exposure (p<0.0001). Furthermore there was a significantly greater deletion frequency in 'usually' sun exposed compared with 'occasionally' sun-exposed body sites in both the dermis (p=0.0018) and epidermis (p<0.0001). Investigation of the 3895 bp deletion in the same NMSC samples used in a previous study of the 4977 bp common deletion, showed a greater frequency of the 3895 bp deletion (8/10 vs 4/10, respectively). Additionally, we have linked the 3895 bp deletion with the UVR component of sunlight by inducing the deletion in vitro with repetitive sub-lethal doses of a UVA+UVB light source.

Conservation genetics of the east Pacific green turtle (Chelonia mydas) in Michoacan, Mexico.

The main continental nesting rookeries of the east Pacific green turtle (EPGT), Chelonia mydas, on the Michoacan (Mexico) coast suffered drastic population declines following intense exploitation in the 1960s--1970s with annual abundance of nesting females plummeting from about 25,000 to an average of about 1400 between 1982 and 2001. Analyses of data from three nDNA microsatellite loci and 400 bp mtDNA control region sequences from a total of 123 nesting females sampled from four Michoacan rookeries found no evidence of population sub-structuring. The recent order of magnitude reduction in the population size shows no apparent impact on genetic diversity in either control region sequences (overall h = 0.48; pi = 0.0036) or microsatellite loci (overall Na = 20.8; Hexp = 0.895). Our estimates of annual effective female population size (Nef; from theta = 2Nemicron) of 1.9-2.3 x 10(3), in spite of being an order of magnitude below historical records, appear to be sufficient to allow recovery of this population without significant loss of genetic diversity. These findings highlight the importance of continued conservation to reverse the decline of this population before it becomes vulnerable to genetic erosion.

Large-scale mitochondrial DNA deletion mutations and nuclear genome instability in human breast cancer

Deletion mutations in mitochondrial (mt) DNA (mtDNA) as well as microsatellite instability (MSI) and loss of heterozygosity (LOH) in nuclear DNA (nDNA) exist in human cancer. We determined if: (1) large-scale mtDNA deletion mutations were present in cancerous and not in normal breast tissue, and (2) combining mt- and nDNA findings would provide complementary information to identify breast cancer. Thirty-nine matched breast cancer/histologically normal and 23 “true” normal tissue samples from women without breast cancer were microdissected and DNA extracted. 4977, ∼3938, ∼4388 and ∼4576 bp deletions were observed, with the 4576 bp deletion being present in 0% of true normal, 13% of histologically normal specimens from a cancerous breast and 77% of breast cancers. The other three deletions were not specific to a breast containing cancer. LOH was found in 66.7% and MSI in 38.5% of samples. 38/39 (97.4%) tumors had at least one nDNA or 4576 bp mtDNA alteration, suggesting that mt- and nDNA analysis provides complementary information in breast cancer detection. The 4576 bp deletion appears to indicate cancer in the breast. The higher mtDNA copy number in cancer coupled with a mtDNA deletion mutation which appears specific for breasts which contain cancer may prove to be a good target to screen for cancer in the breast, including specimens of low and/or mixed cellularity.

Mitochondrial DNA-deletions in women with a history of a trisomy-21 pregnancy

Objective: To investigate whether damage by reactive oxygen species (ROS) is related to the incidence of Down’s syndrome pregnancies. Design: Case-control study. Cases: women with a history of a Down’s syndrome pregnancy (N=30), controls: women who gave birth of at least two children and without a history of any Down’s syndrome affected pregnancies (N=30). Materials/Methods: A specific mitochondrial DNA deletion (’common’ 4977 bp deletion), indicating past exposition to ROS, was measured in buccal mucosa cells and peripheral lymphocytes from all participants. The incidence of the deletion was compared between the two groups using a chi-square test. Results: No common deletions could be detected in leukocyte DNA. In buccal mucosa cells, the common deletion was present in 20% of the control group (6 out of 30) whereas is was absent in the Down’s syndrome group (0 out of 30), p <0.05. Conclusions: This finding lends no support to the hypothesis that ROS related ovarian ageing, measured by the amount of 4977 bp mtDNA deletions, plays an important role in the cause of Down’s Syndrome. To reach a definitive conclusion however, the amount of mtDNA mutations should be measured in ovarian tissue. Supported by: The Jan Dekkerstichting and Dr Ludgardine Bouwmanstichting, Amsterdam, The Netherlands.

Social system of the Alaotran gentle lemur (Hapalemur griseus alaotrensis): genetic characterization of group composition and mating system.

Group composition and mating system were investigated in wild Alaotran gentle lemurs (Hapalemur griseus alaotrensis) using genetic markers. These small-bodied herbivorous and cathemeral primates are endemic to the reed and papyrus beds around Lac Alaotra in Madagascar. They live in small groups in small, defended territories. Data were collected during the rainy seasons in 1996, 1997, and 1999, and include 99 individuals from 22 neighboring social groups and an additional 30 animals from other areas representing most of the geographic range. Animals were located by researchers canoeing in the marshes bordering the lake. After a group's size was determined by direct observation, all individuals were captured, marked, and released. During handling each individual was weighed and sexed, and hair samples for DNA extraction were collected. A 342 bp mtDNA control region sequence and 10 nuclear microsatellite loci provided multilocus genotypes that were used to assess pedigree relations and population structure. Alaotran gentle lemurs were found in groups of two to nine individuals (mean: 4.3), comprising one or two breeding females, their offspring, and one reproducing male. Solitary individuals of both sexes were encountered rarely. Breeding females were the permanent core of the social groups, whereas intergroup transfer of reproductive males was relatively frequent. Forty percent of all reproducing groups contained two breeding females, which were related to each other as closely as mother-daughter or full sisters. Parentage assessment revealed a variable mating system ranging from serial monogamy to polygyny within social groups. At least 8% of paternities involved extragroup males. Additional data on life history and reproduction are presented, and the social system of the Alaotran gentle lemur is discussed in the light of the new genetic findings.

History and integrity of thoroughbred dam lines revealed in equine mtDNA variation.

Mitochondrial DNA (mtDNA) D-loop sequences (381 bp) from 100 thoroughbreds in 19 of the most common matrilineal female families were used to reconstruct a founder female population for the thoroughbred ( approximately 1650-1750 AD). Seventeen haplotypes were found to have contributed to the 19 female lineages. In order to place the reconstructed founder population in wider historical context, we examined, using both single strand conformation polymorphism and direct sequence analysis, variation in a 343 bp mtDNA fragment in that population and 13 other horse populations of disparate provenance. Interpopulation diversity analyses revealed no significant difference in variation between the thoroughbred founder population and the 13 other diverse horse populations and suggested a non-random partitioning of diversity among geographically diverse horse populations. Within thoroughbreds, almost half of the female families, which are each considered from pedigrees to have matrilines converging to one of >30 historically recognized female ancestors, contained sequences which were inconsistent with common descent. Examination of the anomalies in the context of pedigree records suggests the majority might be best explained as confusion of mares at the foundation stages, although some could have some bearing on more recent (19th century - 1980) lineages. We have used this combination of molecular and historical information to identify some of the founder dams and to make new interpretations about the early history of the thoroughbred.

Mitochondrial DNA mutations in the parotid gland of cigarette smokers and non-smokers

It has previously been demonstrated that mitochondrial DNA (mtDNA) mutations accumulate in the lung and increase in frequency with age. It has also been shown that the level of mtDNA mutations including deletions and base substitutions are elevated in lung tissue of smokers relative to non-smokers. We have previously shown that the ‘common’ 4977 bp mtDNA deletion is present in the parotid (salivary) gland of smokers and non-smokers and that there is a significant increase in the level of this deletion in Warthins tumour, an oncocytoma of the parotid gland. In this study we used semi-quantitative PCR to confirm the presence of 4977 bp mtDNA deletion in the parotid gland of non-smokers and smokers. Importantly, we show that the deletion accumulates with age regardless of smoking status and that there was no significant difference in the level of the 4977 bp deletion in parotid tissue of smokers and non-smokers. Using strand conformational polymorphism (SSCP) and direct sequencing we also found 5/23 smokers had parotid tissue specific base substitutions: either an A/T to G/C transition at A4767 or a G/C to A/T transition at G4853. These results are evidence of age related increase in the 4977 bp deletion and a higher level of mutations, probably due to oxidative damage, in the parotid gland of smokers.

Depletion of mitochondrial DNA in the skeletal muscle of two cirrhotic patients with severe asthenia

Qualitative and quantitative alterations of mitochondrial DNA (mtDNA) in the skeletal muscle from two patients with cirrhosis and severe asthenia have been studied. The 4977 bp (mtDNA4977) and the 7436 bp (mtDNA7436) mtDNA deletions, as well as other mtDNA deletions, revealed by long extension PCR (LX-PCR), were found in the two patients, whereas the 10,422 bp (mtDNA10,422) mtDNA deletion was absent. Altogether, the qualitative alterations of mtDNA in cirrhotic patients with severe asthenia were comparable to those of age-matched healthy individuals. The mtDNA content, on the contrary, was substantially decreased in both patients with respect to control. Such mtDNA depletion might be explained by an increased, disease-related, oxidative damage to mtDNA, which probably affects the replication of the mitochondrial genome as already suggested in other oxidative stress-associated diseases.

Large-scale mitochondrial DNA deletions in skeletal muscle of patients with end-stage renal disease

End-stage renal disease (ESRD) is associated with enhanced oxidative stress. This disease state provides a unique system for investigating the deleterious effect of exogenous sources of free radicals and reactive oxygen species (ROS) on mitochondrial DNA (mtDNA). To test the hypothesis that uremic milieu might cause more severe damage to mtDNA, we investigated the prevalence and abundance of mtDNA deletions in the skeletal muscles of ESRD patients. The results showed that the frequencies of occurrence of the 4977 bp and 7436 bp deletions of mtDNA in the muscle tissues of the older ESRD patients were higher than those of the younger patients. The frequency of occurrence of the 4977 bp-deleted mtDNA in the muscle was 33.3% for the patients in the age group of < 40 years, 66.6% in the 41–60-year-old group, 100% in the 61–80-year-old group, and 100% in patients >80 years of age, respectively. Only 22% of the normal aged controls carried the 4977 bp mtDNA deletion, whereas 77% (17/22) of the ESRD patients exhibited the mtDNA deletion. Using a semiquantitative PCR method, we determined the proportion of the 4977 bp-deleted mtDNA from the muscles that had been confirmed to harbor the deletion. We found that the proportions of the 4977 bp-deleted mtDNA in the muscle were significantly higher than those of the aged matched controls. Using long-range PCR techniques, a distinctive array of mtDNA deletions was demonstrated in the muscle of uremic patients. In summary, we found diverse and multiple mtDNA deletions in the skeletal muscles of ESRD patients. These deletions are more prevalent and abundant in ESRD patients than those found in normal populations. Accumulation of uremic toxins and impaired free radical scavenging systems may be responsible for the increased oxidative stress in ESRD patients. Such stress may result in oxidative damage and aging-associated mutation of the mitochondrial genome.

Clinical and genetic studies of Japanese homozygotes for the Gaucher disease L444P mutation.

In patients originally genotyped as homoallelic for the Gaucher disease (GD) L444P (1448C) mutation, we sought to confirm previously reported phenotypic differences between Caucasians and Japanese, to determine the prevalence and phenotypic impact of recombinant alleles, and to explore the phenotypic influence of genetic background. We therefore analyzed data from longer-term clinical follow-up, more comprehensive genotyping and polymorphism and mitochondrial DNA (mtDNA) testing in all known Japanese L444P homozygotes (n=15). Our studies demonstrated that, of 12 patients in our series originally diagnosed with non-neuronopathic GD, 9 developed neurological signs/symptoms during follow-up (at a mean of 14 years 11 months +/- 11 years 4 months). Of three patients originally diagnosed with acute neuronopathic (type 2) GD, all three were compound heterozygotes for L444P and the complex allele RecNci I. In the entire series, Pvu II and liver erythrocyte pyruvate kinase (PKLR) polymorphism and prevalence of the 9 bp mtDNA deletion were heterogeneous, and these background genetic factors could not predict phenotypic expression. Our data suggest that, in Japanese as in Caucasian patients, the L444P/L444P genotype is highly associated with subacute neuronopathic (type 3) GD, and the presence of a complex allele together with an L444P allele leads to type 2 disease. Our findings also underline the importance of comprehensive genotyping (particularly testing for recombinant alleles), long-term follow-up and careful neurological examination in patients with early-onset GD. Such measures ultimately may improve genotype/phenotype correlations and, with them, genetic counseling and therapeutic decision making.

Mitochondrial DNA deletions are associated with ischemia and aging in Balb/c mouse brain

Deletions in the mitochondrial DNA (mtDNA) of Balb/c mouse cerebrums, resembling deletions found in elderly humans or in patients with certain disorders, were detected by PCR. Analysis was carried out on mice of various ages and on mice in which the bilateral common carotid arteries had been incompletely ligated to reconstruct cerebral ischemia. A 3,867 bp mtDNA deletion was present only in old or ischemic mouse groups. Among the non-ischemic groups, it was found in 0 of 12 weaning, 0 of 12 young, and four of eight old mice. Among the ischemic groups, it was found in 12 of 17 young and 11 of 11 old mice. Moreover, the percentage of total mtDNA containing deletions was 22% for the old non-ischemic group, 37% for the young ischemic group, and 69% for the old ischemic group. In addition, PCR analysis detected two other deletions of 3,726 bp and 4,236 bp in 4 of the 11 old ischemic cerebrums. The results indicate that mtDNA deletions are associated with aging, that ischemia increases the incidence of mtDNA deletions, and that mtDNA deletions resulting from ischemia are more likely to occur in old mice than in young mice.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817).

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species' range. Eighteen different haplotypes were defined in the approximately 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 +/- 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.

Skeletal muscle mitochondrial DNA injury in patients with unilateral peripheral arterial disease.

Patients with peripheral arterial disease (PAD) have exercise limitation due to claudication-limited pain and metabolic alterations in skeletal muscle. PAD is also associated with oxidative stress, which is a known cause of mitochondrial DNA (mtDNA) injury. The present study was designed to test the hypothesis that PAD is associated with mtDNA injury, as reflected by an increased frequency of a specific 4977-base pair (bp) mtDNA deletion mutation. The deletion frequency was quantified in gastrocnemius muscle of 8 patients with unilateral PAD and 10 age-matched control subjects with the use of polymerase chain reaction methodologies. Muscle from the hemodynamically unaffected (less affected) PAD limb showed an 8-fold increased deletion frequency and the hemodynamically affected (worse affected) PAD limb had a 17-fold increased deletion frequency compared with muscle from control subjects. The frequency of the 4977-bp deletion in the worse-affected limb was positively correlated with the age of the patients but not the claudication-limited exercise performance of the patients. Total mtDNA content, citrate synthase activity, and cytochrome c oxidase activity were not different in the muscle from the 3 limb populations. However, the ratio of citrate synthase to cytochrome c oxidase was higher in the worse- versus less-affected limbs of PAD patients. The present study demonstrates a large increase in the frequency of the mtDNA 4977-bp deletion in patients with PAD but in a distribution not limited to the hemodynamically affected limb.

Smoking-associated mitochondrial DNA mutations and lipid peroxidation in human lung tissues.

To investigate the effect of cigarette smoking on mitochondrial DNA (mtDNA) mutation and lipid peroxidation in lung tissues, 152 samples from lung resections were collected. A novel deletion of 4,839 bp of mtDNA was found in 80 (52.6%) of the 152 lung samples. The breakpoints of the 4,839-bp mtDNA deletion were flanked by a nine-nucleotide direct repeat (5'-CATACACAA-3'). The frequency of occurrence and the proportion of the 4,839-bp mtDNA deletion in the lung increased significantly with the smoking index in terms of pack-years (P < 0.05). The incidence and proportion of the 4,839-bp mtDNA deletion in the lung tissues of current smokers were significantly higher than in those of nonsmokers (P < 0.05). In addition, we found that the content of lipid peroxides in the lung tissues of the smokers was significantly higher than in that of nonsmokers, and increased with the smoking index. The average malondialdehyde level in the lung tissues was 12.81 +/- 4.99 micromol/g for subjects with a smoking index of more than 50 pack-yr, and was 5.39 +/- 0.48 micromol/g for nonsmokers (P < 0.05). Multiple regression analysis showed that the smoking index, tissue lipid-peroxide content, and FEV1/FVC ratio were correlated with the proportion of the 4,839-bp mtDNA deletion in the lung. These results suggest that cigarette smoke plays an important role in the increase in mtDNA mutation and lipid peroxidation in the lung tissues of smokers.

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240-bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.

Detection of the age-dependent 4977 bp deletion of mitochondrial DNA. A pilot study.

In recent years a number of mitochondrial DNA (mtDNA) deletions have been detected in various tissues from individuals over 20 years of age. It has been postulated that these deletions are associated with natural aging. In order to determine whether a correlation exists between age and the amount of deleted 4977 bp mtDNA, we used two PCR reactions to study total DNA (nuclear and mitochondrial DNA) extracted from skeletal muscle (m. iliopsoas) obtained at autopsy from 93 individuals representing a wide age spectrum (range: 3 months-97 years). The primer pair L15/H15 was used to amplify a 533 bp fragment of intact mtDNA to determine the percentage of total DNA. A second PCR with the primer pair L35/H35 was then employed to amplify a 667 bp fragment of the deleted mtDNA. The amount of template DNA necessary to amplify the specific fragments of deleted mtDNA was found to decrease with age. Whereas no 4977 bp deletion could be detected in subjects under 20 years of age even with 1000 ng of total DNA, in individuals aged 21 to 30 years 1000 ng total DNA were sufficient. Only 1 ng total DNA was needed in all individuals over 70. Our results show that the 4977 bp deletion can be a useful marker of natural aging in human subjects.

Method for in situ investigation of mitochondrial DNA deletions

A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells. Hum Mutat 10:489–495, 1997. © 1997 Wiley-Liss, Inc.