Detection of G1138A Mutation of the FGFR3 Gene in Tooth Material from a 180-Year-Old Museological Achondroplastic Skeleton

Contains fulltext : 189828.pdf (publisher's version ) (Open Access

Analysis of age-dependent DNA methylation changes in plucked hair samples using massive parallel sequencing

<jats:title>Abstract</jats:title><jats:p>The analysis of age-dependent DNA methylation changes is a valuable tool in epigenetic research and forensic genetics. With some exceptions, most studies in the past concentrated on the analysis of blood, buccal, and saliva samples. Another important sample type in forensic investigations is hair, where age-dependent DNA methylation has not been investigated so far. In this pilot study a deeper look was taken at the possibilities and challenges of DNA methylation analysis in hair. The DNA methylation of selected age-dependent 5’-C-phosphate-G‑3’ (CpG) sites were characterized for their potential use as a biomarker for age prediction using plucked hair samples and massive parallel sequencing. Plucked hair roots of 49 individuals were included in the study. The DNA methylation of 31 hairs was successfully analyzed. The DNA methylation pattern of 10 loci, including <jats:italic>ELOVL2</jats:italic>, <jats:italic>F5, KLF14</jats:italic>, and <jats:italic>TRIM59</jats:italic>, was determined by amplicon-based massive parallel sequencing. Age-dependent changes were found for several markers. The results demonstrate the possible use of already established age-dependent markers but at the same time they have tissue/cell type-specific characteristics. Special challenges such as low amounts of DNA and degraded DNA as well as the possible heterogeneous cellular composition of plucked hair samples, have to be considered.</jats:p&gt

Melt curves and RMSE values for animal-family specific PCR and HRM of the Phasianidae.

<p>A) Normalized HRM results for 12S rRNA. B) RMSE value range for the comparison of all curves presented in A. C) Normalized HRM results for cytb. D) RMSE value range for the comparison of all curves presented in C. Lower values were calculated for samples of the same species within 12S rRNA and species differentiation is possible. Analyzing cytb only would lead to more inconclusive results, although a slightly different melting behavior is also visible.</p

High-Resolution Melting of 12S rRNA and Cytochrome <i>b</i> DNA Sequences for Discrimination of Species within Distinct European Animal Families

<div><p>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 <i>b</i>. 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.</p></div

Mixture analysis within and between animal groups.

<p>A) Mixtures of pig and human DNA representing animals of two different groups can be successfully resolved. B–D) Mixtures between samples of the same animal group i.e. family. The success of mixture resolution varied between the animal groups.</p

Melt curves and RMSE values for animal-family specific PCR and HRM of the Leporidae.

<p>A) Normalized HRM results for 12S rRNA. The SNP represents an A>G transition. B) RMSE value range for the comparison of all curves presented in A. C) Normalized HRM results for cytb. D) RMSE value range for the comparison of all curves presented in C. Lower values were calculated for samples of the same species and species differentiation is possible. The SNP does not fit into the range obtained for the 12S rRNA rabbit fragments, but does not interfere with the hare curves.</p

Summary of possible visual discriminations within the animal groups analyzed.

<p>Except for the pairs roe deer/red deer and beech marten/otter, discrimination was always possible by the analysis of at least one gene. Within the Mustelidae, the discrimination relies mostly on 12S rRNA, as no successful amplification for cytb was possible for all species.</p

Normalized melt curves using the universal 12S rRNA primers.

<p>The curves of two runs (necessary due to sample number) were merged. A distribution of the T_ms over 3°C can be observed as well as special melt curve features resulting in direct discrimination, e.g. sheep. Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115575#pone.0115575.s002" target="_blank">S2 Fig.</a></p

Mitochondrial DNA control region variation in a population sample from Thailand

Mitochondrial DNA (mtDNA) control region sequences from hair samples of 213 individuals from Thailand were analyzed using Sanger sequencing. A total of 170 different haplotypes were identified, of which 146 occurred only once (unique haplotypes). The dataset showed a random match probability of 0.87% and a haplotype diversity of 0.9960. The samples were assigned to 85 different haplogroups with B5a, F1a1a, and M being the most frequent ones. Pairwise

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