Historical DNA (hDNA), obtained from museum and herbarium specimens, has yielded spectacular new insights into the history of organisms. This includes documenting historical genetic erosion and ...extinction, discovering species new to science, resolving evolutionary relationships, investigating epigenetic effects, and determining origins of infectious diseases. However, the development of best-practices in isolating, processing, and analyzing hDNA remain under-explored, due to the substantial diversity of specimen preparation types, tissue sources, archival ages, and collecting histories. Thus, for hDNA to reach its full potential, and justify the destructive sampling of the rarest specimens, more experimental work using time-series collections, and the development of improved methods to correct for data asymmetries and biases due to DNA degradation are required.
The study of historical DNA (hDNA), utilizing museum specimens, has emerged as a sub-discipline distinct from ancient DNA.Recent advances in hDNA extraction and sequencing support minimally destructive sampling requests for well-represented specimens, and can now be considered routine/low risk.Availability of reference genomes, improved mapping techniques, and decreased costs make whole genome resequencing an attractive option for future hDNA studies.New research is needed to describe and correct for hDNA degradation over decadal-scales, and across diverse tissues and voucher types. This will improve hDNA source selection, and enhance integration with modern DNA. Improved methods are needed for formalin-fixed specimens.Together, modern and hDNA have the potential to accelerate comparative biological research on a scale not seen since museum collections were first established.
The QIAGEN Investigator
Quantiplex
Pro Kit is a real-time quantitative PCR assay utilized by forensic DNA laboratories to determine the amount of amplifiable human and male DNA in a sample prior to ...downstream amplification of specific STR markers for human identity testing. This quantification method includes two internal controls that assist the analyst in a preliminary evaluation of the sample in regard to both inhibition or degradation that may be present in the sample and subsequently affect the more targeted downstream amplification of specific markers for identity testing. The internal controls are analogous to the quality sensors contained in QIAGEN's Investigator
24plex line of amplification kits, ensuring that the sample's performance in the quantitation step can accurately predict the success of the STR amplification results. This chapter describes the physical plate setup of a quantitative PCR assay utilizing the QIAGEN Investigator
Quantiplex
Pro Kit as well as the steps and software configurations involved in running such a plate on the Applied Biosystems 7500 Real-Time PCR System for Human Identification using HID Real-Time PCR Analysis Software v1.1 or 1.2.
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•First report on autoclave assisted green synthesis of AgNPs using neem gum.•Carbonyl, hydroxyl and amino group of gum stabilize AgNPs.•Monodispersed-spherical shaped AgNPs found to ...be <30nm.•Antibacterial mechanism of AgNPs was also proposed.
A simple method for the green synthesis of silver nanoparticles (AgNPs) using autoclave assisted gum extract of neem (Azadirachta indica) has been investigated for the first time. Silver nanoparticles were formed due to reduction of silver nitrate solution when mixed with the gum extract after autoclaving at 121°C and 15psi. The UV–vis absorption spectrum of the biologically reduced reaction mixture showed the surface plasmon peak at 418nm which is characteristic peak of silver nanoparticles. The functional biomolecules present in the gum extract and the interaction between the nanoparticles were identified by the Fourier transform infrared spectroscopy (FTIR) analysis. Average diameter of the synthesized nanoparticles was found to be <30nm, as revealed from transmission electron microscopy (TEM) and atomic force microscopy (AFM) analysis. X-ray diffraction (XRD) analysis confirmed the face-centered cubic crystalline structure of metallic silver. The synthesized silver nanoparticles exhibited antibacterial activity against clinical isolates of Salmonella enteritidis and Bacillus cereus. Moreover, the antibacterial activity of the silver nanoparticles was further confirmed by degradation of test bacterial DNA. The results suggest that the gum mediated synthesized silver nanoparticles could be used as a promising antibacterial agent against clinical pathogens.
We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after ...bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.
•Short-term bloodstain age was estimated by the proportions of long DNA fragments.•Nanopore sequencing was applied to measure DNA fragment length.•The effect of water on DNA degradation was examined using bloodstains wetted once.
•Review on the advantages and drawbacks of current authentication techniques.•Review on the effects of DNA degradation and PCR inhibitors for DNA extraction and analysis.•Review on techniques for ...on-site diagnosis, high-throughput species identification and quantification.
Authentication of food or food supplements with medicinal values is important to avoid adverse toxic effects, provide consumer rights, as well as for certification purpose. Compared to morphological and spectrometric techniques, molecular authentication is found to be accurate, sensitive and reliable. However, DNA degradation and inclusion of inhibitors may lead to failure in PCR amplification. This paper reviews on the existing DNA extraction and PCR protocols, and the use of small size DNA markers with sufficient discriminative power for molecular authentication. Various emerging new molecular techniques such as isothermal amplification for on-site diagnosis, next-generation sequencing for high-throughput species identification, high resolution melting analysis for quick species differentiation, DNA array techniques for rapid detection and quantitative determination in food products are also discussed.
Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such ...as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 μM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.
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•The in vitro inhibitory effects of ADG on the proliferation and cell death of Giardia trophozoites.•ADG led to trophozoite shrinkage, membrane rupture and remarkable changes in morphology.•The administration of ADG led to a reduction of cells in the S/G2M phase.•ADG led to the production of excessive intracellular ROS in Giardia trophozoites.
Ancient genome analysis has become an indispensable tool in studies of human population history and evolution since the breakthrough of whole-genome sequencing technology. The problem remains, ...however, that ancient genomes cannot be analyzed without crushing non-small pieces of precious specimens; moreover, in many cases, there is insufficient DNA remaining in the pieces of sample to obtain whole-genome sequences. In previous studies, therefore, a couple of indicators (e.g. racemization ratios) have been proposed to estimate the endogenous DNA in ancient samples. However, these studies have used polymerase chain reaction (PCR) to test whether endogenous DNA remains, but this has proved inadequate because the success or failure of PCR amplification does not necessarily reflect the DNA remaining. To assess the amount of endogenous DNA, we use the ratios of reads generated by next-generation sequencing (NGS) mapped to the human reference genome sequence. We investigated 40 human remains excavated from three shell-mound sites of the late to final Jomon culture. The associations between the environmental/molecular factors and the mapping ratios (MRs) were examined. There were no significant associations between the environmental factors and MRs, or between the collagen residual ratios (CRRs) and the MRs. However, we found a significant association between CRRs in rib bones and MRs. The weight of bone required to measure residual collagen is much less than that required to obtain the DNA necessary for NGS analysis, and the process of measuring CRRs is always involved in dating. Hence, we propose the collagen in the ribs as a good indicator for successful ancient genome analyses.
Abstract Forensic DNA identification techniques are principally based on determination of the size or sequence of desired PCR products. The fragmentation of DNA templates or the structural ...modifications that can occur during the decomposition process can impact the outcomes of the analytical procedures. This study reviews the pathways involved in cell death and DNA decomposition and the subsequent difficulties these present in DNA analysis of degraded samples.
Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental ...degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream‐dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full‐sun and shaded treatments degraded exponentially to <1% of the original concentration after 3 days. eDNA was no longer detectable in full‐sun samples after 8 days, whereas eDNA was detected in 20% of shaded samples after 11 days and 100% of refrigerated control samples after 18 days. When translocated into unoccupied streams, salamanders were detectable after 6 h, but only when densities were relatively high (0.2481 individuals/m2) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 h after introduction, reaching 0.0022 ng/L. Within 1 h of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.
Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report ...that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks. Intriguingly, nascent DNA degradation by the ROS-ATM-MRE11 cascade is also triggered by hypoxia, which elevates signaling-competent ROS and attenuates functional HDR without arresting replication forks. Under these conditions, MRE11 degrades daughter-strand DNA gaps, which accumulate behind active replisomes and attract error-prone DNA polymerases to escalate mutation rates. Thus, HDR safeguards replicating genomes against metabolic assaults by restraining mutagenic repair at aberrantly processed nascent DNA. These findings have implications for cancer evolution and tumor therapy.
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•Homologous recombination repairs replication-born DSGs•Metabolic activation of the ROS-ATM-MRE11 nuclease pathway elongates unrepaired DSGs•Hypoxia enforces accumulation of DSGs and their processing by ROS-ATM-MRE11•Extended DSGs are the key drivers of mutagenesis and genome instability in hypoxia
Somyajit, Spies, et al. elucidate here a direct mechanistic connection among the three major regulatory modules during oxygen starvation: metabolic rewiring, deficiency of homologous recombination, and error-prone DNA synthesis. This link provides insights into the high levels of mutagenesis and genome instability in cells growing in a hypoxic environment.
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