Influence of cyanoacrylate on the efficiency of forensic PCRs.

Cyanoacrylate ester (CA) is commonly used by criminalists to detect latent fingerprints on smooth surfaces. We investigated whether this treatment has an influence on a subsequent DNA typing of biological stains, and on the efficiency of three different forensic PCRs (mtDNA, Y-STR determination and the Profiler Plus kit). Using fluorescence labeled primers and an automated detection system, we could show that the presence of CA led to weaker PCR products. Depending on the DNA extraction method the amplification results were significantly weaker compared to untreated controls. To simulate forensic cases we prepared blood and saliva stains on glass slides, extracted the DNA using two different methods and compared the signal intensities of the amplified DNA fragments. Depending on the extraction methods, the presence of CA significantly hampered the amplification of DNA from small stains whereas there was virtually no difference comparing the amplification results of DNA extracted from bigger stains.

[The mitochondrial genome and aging]. / Das mitochondriale Genom und Altern.

There is a lot of evidence that age-associated alterations of the mitochondrial genome occur, especially in postmitotic tissues such as brain, heart and skeletal muscle. These alterations are supposed to be a result of an attack of free radicals generated as normal byproducts of oxidative phosphorylation and lead to damage of proteins, lipids, and DNA. The alterations of mtDNA include oxidative damage of base pairs, point mutations, large-scale deletions or duplications. The 4977 bp deletion or "common deletion" reveals an age-dependent accumulation in postmitotic tissues, but not in fast-dividing tissues such as blood cells. In addition, it is observed that a tissue-specific accumulation occurs with the highest abundance in the basal ganglia, followed by skeletal muscle, heart, and lowest in cerebellar tissue. Third, pathological alterations of specific tissue, like ischemia/reperfusion events, display a pronounced accumulation of the deletion compared to age-matched controls. Because there are many mtDNA mutations, further analysis of all alterations of mtDNA will elucidate its role in the phenomenon of aging. Despite some criticisms of this free radical theory of aging, there is a lot of experimental evidence to support the important role of mitochondria in organismal aging.

Quantification of mitochondrial DNA in human blood cells using an automated detection system.

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.

Estimation of age at death based on quantitation of the 4977-bp deletion of human mitochondrial DNA in skeletal muscle.

The 4977-bp deletion in human mitochondrial DNA (mtDNA) is known to accumulate in various tissues with age. Since this deletion in mtDNA correlates closest with age in muscle tissue, iliopsoas muscle tissue was taken at autopsy from 50 persons aged 24-97 years to determine whether age at death can be estimated based on the amount of the 4977-bp deletion in skeletal muscle. Total DNA (nuclear and mtDNA) was extracted from 100 mg tissue and the 4977-bp deletion quantified using a kinetic polymerase chain reaction (PCR) followed by visualization of the products on silver stained polyacrylamide gels. The amount of the 4977-bp deletion of mtDNA ranged from 0.00049% to 0.14% depending on age, with a correlation coefficient of r = 0.83 (P = 0.0001). In forensic practice this method can aid in the estimation of age at death with a relatively wide confidence interval, thus enabling a discrimination between young and elderly persons in the identification of human remains based solely on skeletal muscle.

Demonstration of the 4977 bp deletion in human mitochondrial DNA from intravital and postmortem blood.

The 4977 bp deletion in mitochondrial DNA (mtDNA) is known to accumulate with age in various human tissues. Findings regarding its accumulation in blood, however, have so far been contradictory. We investigated the levels of the 4977 bp deletion in mtDNA from 100 intravital and postmortem blood samples. Applying an improved version of a PCR plus silver staining of polyacrylamide gels, we could detect the 4977 bp deletion in blood of healthy individuals over 20 years of age. While the 4977 bp deletion in blood is subject to a certain age dependence, it appears to be influenced by additional factors. A Primer-Shift-Assay amplifying four different deletion-specific fragments showed that the smaller fragments were amplified with a higher amplification efficiency than the larger fragments. The deletion-specific 389 bp fragment was demonstrated in 73% of individuals over 80 years of age, but in only 46% of individuals between 21 and 30 years old whereas the largest 802 bp deletion-specific fragment was detectable in 38% of subjects over 80 years of age, and in only 15% of individuals under 30 years of age. Deletion-specific fragments were not detected in a single individual under 20 years old, nor in fetal blood. In this work, we demonstrate for the first time the detection of 4977 bp specific fragments in blood of healthy individuals without the necessity of using a nested PCR. The deletion is detectable in postmortal and intravital blood, so that the occurrence of the 4977 bp deletion seems to be a physiological and not only a postmortal process.

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.