Source Of DNA Research Articles

Evaluation of low-cost bone and teeth processing methods for automated DNA extraction

Bone and teeth can often be the only, or the best, biological materials available as DNA sources for the identification of decomposed or skeletal human remains. While these hard tissues are resistant to decay and therefore offer endogenous DNA a certain protection from the environment, their mineral matrices make DNA extraction challenging, a problem compounded by the low DNA content and high degradation levels usually associated with these tissues. To help release DNA from the mineral matrix, they are generally ground into a powder to increase their surface area to the extraction reagents. Furthermore, it is now common to perform a demineralization to dissolve the powder and release DNA that remains trapped. A common pulverization method consists of the use of a cryogenic grinder, which prevents heating of the sample and avoids subjecting the DNA to heat damage, but this equipment is costly. That said, little is known about the effect of lower-cost alternatives to the expensive cryogenic grinder on the quality and quantity of DNA recovered. Thus, this study aimed to evaluate inexpensive grinding methods for their potential for use in a forensic laboratory setting. The methods deemed to be viable options for this purpose were investigated to determine their effects on the quantity and quality of extracted DNA. Bone samples were also subjected to freezing prior to pulverization to assess the possibility that a lower sample temperature could limit heat damage to the DNA during processing. Pig bone and tooth powders were extracted using an original and a modified PrepFiler BTA™ Kit protocol, with the latter featuring an added demineralization step. Out of the methods explored, a coffee grinder and mortar and pestle were determined to be the most suitable for bone and teeth processing in a forensic laboratory setting. No significant differences between the two methods were found regarding the quantity and quality of DNA recovered and the rate of powder recovery, although more powder loss occurred when teeth were crushed with the mortar and pestle. Likewise, freezing samples before grinding did not have a significant impact on DNA quality and quantity. Finally, the original DNA extraction protocol performed significantly better than the modified one for the quantity of DNA recovered from both bone and teeth, while the quality was only superior for the bone.

Whole Genome Sequencing in Advanced Lung Cancer can be Performed Using Diff-Quik Cytology Smears Derived from Endobronchial Ultrasound, Transbronchial Needle Aspiration (EBUS TBNA)

IntroductionMaximising alternative sample types for genomics in advanced lung cancer is important because bronchoscopic samples may sometimes be insufficient for this purpose. Further, the clinical applications of comprehensive molecular analysis such as whole genome sequencing (WGS) are rapidly developing. Diff-Quik cytology smears from EBUS TBNA is an alternative source of DNA, but its feasibility for WGS has not been previously demonstrated.MethodsDiff-Quik smears were collected along with research cell pellets.ResultsTumour content of smears were compared to research cell pellets from 42 patients, which showed good correlation (Spearman correlation 0.85, P < 0.0001). A subset of eight smears underwent WGS, which presented similar mutation profiles to WGS of the matched cell pellet. DNA yield was predicted using a regression equation of the smears cytology features, which correctly predicted DNA yield > 1500 ng in 7 out of 8 smears.ConclusionsWGS of commonly collected Diff-Quik slides is feasible and their DNA yield can be predicted.

Differentiation of vaginal cells from epidermal cells using morphological and autofluorescence properties: Implications for sexual assault casework involving digital penetration

Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. To assist the forensic scientist address source and activity level propositions there is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across > 80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving hand swabbings following digital vaginal penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration hand swab samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue (≥ 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types.

A systematic review of noninvasive preimplantation genetic testing for aneuploidy

Non-invasive/minimally invasive PGT-A is a tool that may one day become the gold standard for embryo chromosomal screening. Investigations on this topic have ranged from studying the culture media of embryos to the fluid inside the blastocoel, all in an attempt to find a reliable source of DNA without the need to biopsy the embryo. There is great interest across the board, both from those for and against biopsy, for a reliable test process that would give the patient and provider the same information possible from a biopsy without the risk. We aim to explore the current available research to better understand the utility and accuracy of PGT-A with these new sampling techniques. General concordance rates with comparison to biopsy-based PGT-A are promising, but it is clear that additional research and understanding is needed prior to adopting non-invasive/minimally invasive PGT-A as a widely-used tool with strong clinical utility.

Increased heterozygosity in low-pass sequencing data allows identification of blood chimeras in cattle.

In about 90% of multiple pregnancies in cattle, shared blood circulation between fetuses leads to genetic chimerism in peripheral blood and can reduce reproductive performance in heterosexual co-twins. However, the early detection of heterosexual chimeras requires specialized tests. Here, we used low-pass sequencing data with a median coverage of 0.64× generated from blood samples of 322 F1 crosses between beef and dairy cattle and identified 20 putative blood chimeras through increased levels of genome-wide heterozygosity. In contrast, for 77 samples with routine SNP microarray data generated from hair bulbs of the same F1s, we found no evidence of chimerism, simultaneously observing high levels of genotype discordance with sequencing data. Fifteen out of 18 reported twins showed signs of blood chimerism, in line with previous reports, whereas the presence of five alleged singletons with strong signs of chimerism suggests that the in-utero death rate of co-twins is at the upper limit of former estimates. Together, our results show that low-pass sequencing data allow reliable screening for blood chimeras. They further affirm that blood is not recommended as a source of DNA for the detection of germline variants.

A new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong.

Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.

Molecular Screening of β-Lactoglobulin Gene Variation for Allergy-free Milk Production in Indonesian Friesian Holstein

The most allergenic protein in cow’s milk for new-borns is Beta Lactoglobulin (β-LG), which is also one of the bovine whey proteins. As a result, choosing suitable animals to create free allergenic protein is essential. In this study, Indonesian Friesian Holstein’s genetic variants in the β-LG gene were examined). Forty-two blood samples were sampled as DNA sources. The β-LG gene allele and genotype variant were identified using PCR-RFLP, or Polymerase Chain Reaction - Restriction Fragment Length Polymorphism. Successful amplification of the β-LG gene with a 247 bp product size was followed by DNA sequencing and HaeIII enzyme digestion (GG|CC). There were discovered to be three genotypes and two types of alleles, A and B, with the frequencies being AA (0.26), AB (0.50), and BB (0.24). The population is in equilibrium because A and B have respective allele frequencies of 0.51 and 0.49. In conclusion, molecular-assisted selection can create allergen-free milk by utilizing the genotype variants of β-LG genes.

Deep Domain Adaptation Enhances Amplification Curve Analysis for Single-Channel Multiplexing in Real-Time PCR.

Data-driven approaches for molecular diagnostics are emerging as an alternative to perform an accurate and inexpensive multi-pathogen detection. A novel technique called Amplification Curve Analysis (ACA) has been recently developed by coupling machine learning and real-time Polymerase Chain Reaction (qPCR) to enable the simultaneous detection of multiple targets in a single reaction well. However, target classification purely relying on the amplification curve shapes currently faces several challenges, such as distribution discrepancies between different data sources of synthetic DNA and clinical samples (i.e., training vs testing). Optimisation of computational models is required to achieve higher performance of ACA classification in multiplex qPCR through the reduction of those discrepancies. Here, we proposed a novel transformer-based conditional domain adversarial network (T-CDAN) to eliminate data distribution differences between the source domain (synthetic DNA data) and the target domain (clinical isolate data). The labelled training data from the source domain and unlabelled testing data from the target domain are fed into the T-CDAN, which learns both domains' information simultaneously. After mapping the inputs into a domain-irrelevant space, T-CDAN removes the feature distribution differences and provides a clearer decision boundary for the classifier, resulting in a more accurate pathogen identification. Evaluation of 198 clinical isolates containing three types of carbapenem-resistant genes (blaNDM, blaIMP and blaOXA-48) illustrates a curve-level accuracy of 93.1% and a sample-level accuracy of 97.0% using T-CDAN, showing an accuracy improvement of 20.9% and 4.9% respectively, compared with previous methods. This research emphasises the importance of deep domain adaptation to enable high-level multiplexing in a single qPCR reaction, providing a solid approach to extend qPCR instruments' capabilities without hardware modification in real-world clinical applications.

Rock pools as a source of environmental DNA for the detection of the threatened Pilbara olive python (Liasis olivaceus barroni)

Environmental DNA (eDNA) research is transforming biomonitoring at a global scale, but applicability to reptiles has been restricted because of their presumed low shedding rate. Consequently, eDNA may have considerable limitations as a biomonitoring tool in Australia where 40% of the terrestrial vertebrates are reptiles. However, there is a need to evaluate if method improvements, such as targeting certain substrates, improve the ability to detect reptile eDNA. The Pilbara olive python (Liasis olivaceus barroni) is an uncommon and elusive Australian top predator with a high conservation priority. Like many other snake species, Pilbara olive pythons are challenging to monitor with traditional survey methods; therefore, exploring an eDNA-based approach is highly relevant. The pythons are known to occasionally reside in rock pools. Thus, the development of a reliable eDNA-based approach to detect the pythons in water would provide a needed alternative method. Here, we use a previously developed metabarcoding assay targeting reptiles, to sequence a total of 228 water samples collected from 40 rock pools and drainage pools from six broad locations across the Pilbara region of Western Australia, and we confirm the presence of Pilbara olive python eDNA in 37 samples from 12 of those pools at three of the six broad sampling locations. Other vertebrate taxa, including other reptiles, amphibians, mammals, and birds, were also detected. Our documented ability to detect Pilbara olive python eDNA from rock pool water samples represents an important step toward eDNA-based precision monitoring of this species.

The effect of ctDNA tumor fraction (TF) on overall survival and concordance between tissue genomics and ctDNA in Lung-MAP.

9035 Background: Discordance in genomic profiling between tumor tissue and ctDNA can occur due to inter-patient variability in shed tumor DNA, and intra-patient variability in sources of cell-free DNA (tumor heterogeneity, metastatic variants, CHIP). TF is an aneuploidy-based estimation of tumor-specific DNA in circulation. We evaluated the impact of TF levels on overall survival (OS), mutation concordance and tumor mutation burden in tissue (tTMB) and ctDNA (bTMB) in the LUNGMAP screening study. Methods: Eligible advanced NSCLC pts had blood drawn ≤30 days of fresh tissue biopsy with no intervening therapies. Genomic profiling was done by FoundationOne CDx and FoundationOne Liquid CDx. ctDNA TF was quantified via aneuploidy using deviations in genome-wide SNP coverage or somatic allele frequencies if under aneuploidy LoD. High TF was defined as ≥10%. Concordance among driver genes, cancer genes of interest (CGoI: T P53, PIK3CA, STK11, KEAP1, RB1, PTEN, ATM, BRCA1/2), and variant subtype were assessed by the percentage positive agreement (PPA) and positive predictive value (PPV) with 95% confidence intervals (CI). OS by TF used a Cox model and log-rank test. PPA and TF among CGoIs was compared with a Wilcoxon test. Correlation between tTMB and bTMB used Lin’s coefficient (LC). Results: 167 pts met inclusion criteria; 161 had TF data with 51 (32%) ≥10% (high TF). High TF was associated with worse OS (HR: 2.06 [1.43-2.95], p&lt;0.001). The PPA and PPV ranged from 50%-100% and 60%-100%, respectively for driver genes. The PPA was numerically larger for high versus low TF for most drivers (p=0.18) and significantly larger for all mutations and SNVs combined (Table). For CGoI, the distribution of PPA differed between high and low TF (p=0.03). The median PPA for CGoI (25th%ile,75th%ile) was 56% (50,87) for low TF and 100% (100,100) for high TF. PPV for CGoI did not differ by TF (p=0.20). High TF was associated with higher levels of TMB and bTMB (p&lt;0.01 for both). TMB LC (CI) for low and high TF were 0.46 (0.27,0.61) and 0.69 (0.53,0.81). Conclusions: High TF was associated with worse OS, better PPA among CGoI, and better LC for t/bTMB. TF may improve the clinical utility of mutations detected by liquid biopsies and should be reported and considered in interpreting these results. [Table: see text]

Contaminants reach everywhere: Fish dietary samples should be surface decontaminated prior to molecular diet analysis.

Knowledge of trophic interaction is necessary to understand the dynamics of ecosystems and develop ecosystem-based management. The key data to measure these interactions should come from large-scale diet analyses with good taxonomic resolution. To that end, molecular methods that analyze prey DNA from guts and feces provide high-resolution dietary taxonomic data. However, molecular diet analysis may also produce unreliable results if the samples are contaminated by external sources of DNA. Employing the freshwater European whitefish (Coregonus lavaretus) as a tracer for sample contamination, we studied the possible route of whitefish in beaked redfish (Sebastes mentella) guts sampled in the Barents Sea. We used whitefish-specific COI primers for diagnostic analysis, and fish-specific 12S and metazoa-specific COI primers for metabarcoding analyses of intestine and stomach contents of fish samples that were either not cleaned, water cleaned, or bleach cleaned after being in contact with whitefish. Both the diagnostic and COI metabarcoding revealed clear positive effects of cleaning samples as whitefish were detected in significantly higher numbers of uncleaned samples compared to water or bleach-cleaned samples. Stomachs were more susceptible to contamination than intestines and bleach cleaning reduced the frequency of whitefish contamination. Also, the metabarcoding approach detected significantly more reads of whitefish in the stomach than in intestine samples. The diagnostic analysis and COI metabarcoding detected contaminants in a higher and comparable number of gut samples than the 12S-based approach. Our study underlines thus the importance of surface decontamination of aquatic samples to obtain reliable diet information from molecular data.

Analysis of VNTRs (variable number tandem repeat) D1S80 in Biology students class

DNA can be obtained from all parts of the body with the same profile in each person. The oral mucosal epithelium is one of the DNA sources that is often used for individual identification because of taking it using a swab method that does not hurt the volunteers. The purpose of this study was to determine DNA polymorphism in Biology students of Class of 2022. The sample used in this study were 49 people with details, 34 female and 15 male students. After cheek mucosa was sampling, the PCR and electrophoresis was performed to see the DNA bands formed. The visible band was measured in length and calculated using a formula to see the repeatability of the D1S80 locus on the DNA band. From the 49 samples which were collected, the DNA bands were seen in 27 samples with DNA band lengths ranging from 400-600 bp. The number of bands in each sample were also different. The highest frequency of locus repetitions was 26 repetitions and the lowest was 14 repetitions.

DNA Fingerprinting: A Revolution in the Field of Forensic Sciences

DNA profiling, commonly called DNA fingerprinting or DNA testing, has a wide range of uses, including assisting in criminal cases and wildlife conservation. The firm tissues of the teeth are resistant to external events such as decomposition, bruise, and incineration. Pulp tissue provides a rich DNA source. The review focuses on the various DNA Collection Procedures, the different techniques employed in DNA isolation and amplification. There are a numerous DNA Profiling or Typing systems such as Mitochondrial DNA (mtDNA) analysis, Restriction Fragment Length Polymorphism, Y chromosome analysis, Single Nucleotide Polymorphism (SNP) typing , Short Tandem Repeat (STR) typing, Gender typing, X-chromosome STR Typing. DNA fingerprinting has been applied to solve many cases. Although present DNA analysis techniques are fairly specialized, several upcoming simplifications may increase their simplicity of use.

DNA Methylation: Bridging life’s experience and genetics within forensic scenario

DNA serving as the “blueprints of life” is a complex found in all biological cells carrying the genetic instructions for development and functioning of the organism. Inheritance of unique combination of genetic polymorphism results in DNA profile. In past few decades, the field of forensic genetics has witnessed several advancements from the radio-labelled DNA probes to the Short Tandem Repeats (STR) and Single Nucleotide Polymorphism (SNP) that have fostered the emergence of biological materials as most reliable evidences at a crime scene for the purpose of human identification but then, are inadequate in distinguishing monozygotic twins. Also, these techniques are of unavailing in providing informations such as tissue-specificity of the DNA source, phenotypic identification, and age estimation. Epigenetics, particularly DNA methylation is evolving as a promising technique to overcome such drawbacks of conventional profiling techniques.

Molecular detection of the pathogen of Apis mellifera (Hymenoptera: Apidae) in honey in Indonesia

Abstract. Aditya IGMRA, Purwanto H. 2023. Molecular detection of the pathogen of Apis mellifera (Hymenoptera: Apidae) in honey in Indonesia. Biodiversitas 24: 2612-2622. Apis honey bees play a major role in global crop pollination. The honey bee population faces various threats, one being entomopathogens, responsible for 13% of honey bees' declining population cases. Some major pathogens are Nosema sp., Melissococcus plutonius, and Paenibacillus larvae. Data on the prevalence of these pathogens is not available in Indonesia. Therefore, this study aimed to detect entomopathogen in Java, Bali, and Sumatra honey. We extracted 30 honey samples from Java, Bali, and Sumatra in this study. DNA from honey amplified by conventional Polymerase Chain Reaction (PCR) to detect Nosema apis, Nosema ceranae, M. plutonius, and P. larvae. Amplicons were sequenced to confirm the species of pathogens detected. Results showed that N. ceranae has a 20% prevalence, found in Java and Bali; M. plutonius was, for the first time, found in Indonesia and only in Java, with a 3.33% prevalence. In comparison, this study didn't find N. apis and P. larvae. Environmental factors were studied for their correlation with the incidence of pathogens. From several factors, only precipitation significantly correlates with the occurrence of N. ceranae and M. plutonius, while other factors do not. This research concludes that honey can be used as source of DNA for pathogens detection and monitoring in a certain area globally.

Inhibitors of Polyphosphate and Neutrophil Extracellular Traps.

The contact pathway of blood clotting has received intense interest in recent years as studies have linked it to thrombosis, inflammation, and innate immunity. Because the contact pathway plays little to no role in normal hemostasis, it has emerged as a potential target for safer thromboprotection, relative to currently approved antithrombotic drugs which all target the final common pathway of blood clotting. Research since the mid-2000s has identified polyphosphate, DNA, and RNA as important triggers of the contact pathway with roles in thrombosis, although these molecules also modulate blood clotting and inflammation via mechanisms other than the contact pathway of the clotting cascade. The most significant source of extracellular DNA in many disease settings is in the form of neutrophil extracellular traps (NETs), which have been shown to contribute to incidence and severity of thrombosis. This review summarizes known roles of extracellular polyphosphate and nucleic acids in thrombosis, with an emphasis on novel agents under current development that target the prothrombotic activities of polyphosphate and NETs.

The influence of sample size on two approaches to estimate Black Grouse Lyrurus tetrix population size using non‐invasive sampling methods

Population size is an important parameter to monitor for species conservation and management. This is especially important for rare and endangered species, as declines can give information about anthropogenic impacts and the need for new conservation measures. To estimate population size, various methods of analysis can be used, for which sample size is an important factor. Sample size is particularly important to consider when applying non‐invasive sampling strategies such as sampling faeces or feathers/hairs as a source of DNA, as a means to limit disturbance and stress for the species of concern. We investigated a Black Grouse Lyrurus tetrix population in the eastern part of the Alps, in East Tyrol (Austria), and estimated population size using two approaches: capture–recapture and rarefaction. With a set of 12 polymorphic microsatellite markers, we identified genotypes from faeces and feathers (backed up with 23 tissue samples) and checked for population substructure and gene flow among sampling sites. We estimated population size using four different models from the two approaches (molecular capture–recapture: TIRM, TIRMpart; rarefaction: hyperbolic function – Kohn, exponential function – Eggert). To evaluate the impact of sample size on the estimations, we used the full dataset of 500 samples (‘complete’ dataset) and half the dataset of 250 samples (‘half’ dataset). We also estimated the population size for each sex separately using complete and half datasets to check for sex‐specific differences in population size. We found similar results in three of four models (capture–recapture: capwire TIRM, capwire TIRMpart; rarefaction: rarefaction Kohn). Using just half of the data increased the uncertainties in the estimation of population size in all models used and deviations were particularly large in females, which indicated a sex bias. Only the complete dataset of males had an observation rate of more than two observations/individual, and this observation rate meets the recommendation for using the capwire models. This indicates that, for species with different sex‐specific detectability, larger sample sizes do not generally imply higher observation rates. We conclude that calculating the observation rates and population‐size estimations for each sex separately can improve overall population‐size estimation, especially in species with biased sex ratios and those that exhibit sex‐specific behaviour.

Isolation of salivary cell-free DNA for cancer detection.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

Abstract 2287: Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA

Abstract Purpose: Extracellular vesicles (EVs) have been shown to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform circulating cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated whether EVs could be a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. Methods: evDNA from both DNase-treated and untreated circulating EVs, enriched from patients with advanced pancreatic cancer (n=10), was analyzed by fluorometry and digital droplet PCR to determine the level of evDNA inside and outside (surface-bound) the EVs. To also determine if choice of methodology affected the results, EVs were isolated using four different small EV isolation methods (differential ultracentrifugation, size-exclusion chromatography, affinity purification and precipitation) and differential centrifugation for isolation of large EVs. Western blotting, dynamic light scattering and transmission electron microscopy confirmed the enrichment of EVs. Results: Our results indicated that EVs enriched from patient blood samples contained significantly lower amounts of DNA compared to cfDNA (p &amp;lt; 0.001); &amp;gt;50-fold lower for untreated sEVs and &amp;gt;1000-fold lower for DNase-treated EVs. Thus, most of the detected vesicle-associated DNA seemed to be located outside of the vesicles. Further, tumor-derived DNA in EVs was barely detectable and the tumor-derived fraction in all evDNA samples was similar to that of cfDNA. Conclusion: Our results demonstrated that evDNA do not add any extra information compared with cfDNA as a source of tumor-derived DNA, at least not in patients with advanced pancreatic cancer. Citation Format: Morten Lapin, Kjersti Tjensvoll, Karoline Nedrebø, Eline Taksdal, Hans Janssen, Bjørnar Gilje, Oddmund Nordgard. Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2287.

Infectious disease in the Pleistocene: Old friends or old foes?

The impact of endemic and epidemic disease on humans has traditionally been seen as a comparatively recent historical phenomenon associated with the Neolithisation of human groups, an increase in population size led by sedentarism, and increasing contact with domesticated animals as well as species occupying opportunistic symbiotic and ectosymbiotic relationships with humans. The orthodox approach is that Neolithisation created the conditions for increasing population size able to support a reservoir of infectious disease sufficient to act as selective pressure. This orthodoxy is the result of an overly simplistic reliance on skeletal data assuming that no skeletal lesions equated to a healthy individual, underpinned by the assumption that hunter-gatherer groups were inherently healthy while agricultural groups acted as infectious disease reservoirs. The work of van Blerkom, Am. J. Phys. Anthropol., vol. suppl 37 (2003), Wolfe et al., Nature, vol. 447 (2007) and Houldcroft and Underdown, Am. J. Phys. Anthropol., vol. 160, (2016) has changed this landscape by arguing that humans and pathogens have long been fellow travelers. The package of infectious diseases experienced by our ancient ancestors may not be as dissimilar to modern infectious diseases as was once believed. The importance of DNA, from ancient and modern sources, to the study of the antiquity of infectious disease, and its role as a selective pressure cannot be overstated. Here we consider evidence of ancient epidemic and endemic infectious diseases with inferences from modern and ancient human and hominin DNA, and from circulating and extinct pathogen genomes. We argue that the pandemics of the past are a vital tool to unlock the weapons needed to fight pandemics of the future.