Jana Naue

Evidence for frequent and tissue-specific sequence heteroplasmy in human mitochondrial DNA.

Mitochondrial point heteroplasmy is a common event observed not only in patients with mitochondrial diseases but also in healthy individuals. We here report a comprehensive investigation of heteroplasmy occurrence in human including the whole mitochondrial control region from nine different tissue types of 100 individuals. Sanger sequencing was used as a standard method and results were supported by cloning, minisequencing, and massively parallel sequencing. Only 12% of all individuals showed no heteroplasmy, whereas 88% showed at least one heteroplasmic position within the investigated tissues. In 66% of individuals up to 8 positions were affected. The highest relative number of heteroplasmies was detected in muscle and liver (79%, 69%), followed by brain, hair, and heart (36.7%-30.2%). Lower percentages were observed in bone, blood, lung, and buccal cells (19.8%-16.2%). Accumulation of position-specific heteroplasmies was found in muscle (positions 64, 72, 73, 189, and 408), liver (position 72) and brain (partial deletion at position 71). Deeper analysis of these specific positions in muscle revealed a non-random appearance and position-specific dependency on age. MtDNA heteroplasmy frequency and its potential functional importance have been underestimated in the past and its occurrence is ubiquitous and dependent at least on age, tissue, and position-specific mutation rates.

Factors affecting the detection and quantification of mitochondrial point heteroplasmy using Sanger sequencing and SNaPshot minisequencing

Mitochondrial DNA analysis plays an important role in forensic science as well as in the diagnosis of mitochondrial diseases. The occurrence of two different nucleotides at the same sequence position can be caused either by heteroplasmy or by a mix of samples. The detection of superimposed positions in forensic samples and their quantification can provide additional information and might also be useful to identify a mixed sample. Therefore, the detection and visualization of heteroplasmy has to be robust and sensitive at the same time to allow for reliable interpretation of results and to avoid a loss of information. In this study, different factors influencing the analysis of mitochondrial heteroplasmy (DNA polymerases, PCR and sequencing primers, nucleotide incorporation, and sequence context) were examined. BigDye Sanger sequencing and the SNaPshot minisequencing were compared as to the accuracy of detection using artificially created mitochondrial DNA mixtures. Both sequencing strategies showed to be robust, and the parameters tested showed to have a variable impact on the display of nucleotide ratios. However, experiments revealed a high correlation between the expected and the measured nucleotide ratios in cell mixtures. Compared to the SNaPshot minisequencing, Sanger sequencing proved to be the more robust and reliable method for quantification of nucleotide ratios but showed a lower detection sensitivity of minor cytosine components.

High-resolution melting of 12S rRNA and cytochrome b DNA sequences for discrimination of species within distinct European animal families.

The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM) of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible “modular” manner depending on the question to be solved.

Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment.

Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

Bite through the tent

The authors report on a young boy who was bitten into his face by an unknown animal while being asleep in a tent. Given the bite marks and the location of the scene, members of the mustelidae and canidae families were the first “suspects.” Deoxyribunucleic acid (DNA) recovered from the tent’s wall was analyzed with regard to parts of the mitochondrial 12S ribosomal ribunucleic acid (12S rRNA) and cytochrome b (cytb) genes as well as nuclear short tandem repeats (STRs). Since Sanger sequencing revealed a mixed sequence with a strong human component overlying the nonhuman contributor, an animal screening using a duplex real-time polymerase chain reaction (PCR) with an intercalating dye and melt curve analysis was employed. The results were later confirmed by cloning. The applied commercial canine STR kit verified the animal family (canidae) but did not help in discriminating the species due to cross-species amplification. In the presented case, the real-time PCR assay offered the cheapest and fastest method for animal family determination, which then allowed for an appropriate and sample-saving strategy to characterize the causative animal species.

Modular real-time PCR screening assay for common European animal families

A screening assay based on real-time PCR and melt curve analysis was developed to detect DNA from nine common European animal families/species and human. The assay consists of a 10-cycle universal pre-amplification followed by specific nested PCR and was designed to exploit the different melting temperatures (Tm) of family/species-specific 12S ribosomal ribonucleic acid and cytochrome b fragments, which are amplified in duplex reactions. Case-related modular application is possible. Beyond determination of the animal family and discrimination from human DNA, evaluation of the melt curve in some cases additionally allows for species determination (e.g. cat vs. lynx). The method presents a quick, flexible and sample-saving approach to assess non-human DNA at low expenses, and it is especially useful in resolution of DNA mixtures.

Proof of concept study of age-dependent DNA methylation markers across different tissues by massive parallel sequencing

The use of DNA methylation (DNAm) for chronological age determination has been widely investigated within the last few years for its application within the field of forensic genetics. The majority of forensic studies are based on blood, saliva, and buccal cell samples, respectively. Although these types of samples represent an extensive amount of traces found at a crime scene or are readily available from individuals, samples from other tissues can be relevant for forensic investigations. Age determination could be important for cases involving unidentifiable bodies and based on remaining soft tissue e.g. brain and muscle, or completely depend on hard tissue such as bone. However, due to the cell type specificity of DNAm, it is not evident whether cell type specific age-dependent CpG positions are also applicable for age determination in other cell types. Within this pilot study, we investigated whether 13 previously selected age-dependent loci based on whole blood analysis including amongst others ELOVL2, TRIM59, F5, and KLF14 also have predictive value in other forensically relevant tissues. Samples of brain, bone, muscle, buccal swabs, and whole blood of 29 deceased individuals (age range 0-87 years) were analyzed for these 13 age-dependent markers using massive parallel sequencing. Seven of these loci did show age-dependency in all five tissues. The change of DNAm during lifetime was different in the set of tissues analyzed, and sometimes other CpG sites within the loci showed a higher age-dependency. This pilot study shows the potential of existing blood DNAm markers for age-determination to analyze other tissues than blood. We identified seven known blood-based DNAm markers for use in muscle, brain, bone, buccal swabs, and blood. Nevertheless, a different reference set for each tissue is needed to adapt for tissue-specific changes of the DNAm over time.