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Electrospun cellulose adsorbents are an emergent class of materials applied to a variety of bioprocess separations as an analogue to conventional packed bed chromatography. Electrospun adsorbents have proven to be effective as rapid cycling media, enabling high throughput separation of proteins and viral vectors without compromising selectivity and recovery. However, there is a current lack of knowledge in relation to the manipulation and control of electrospun adsorbent structure with function and performance to cater to the separation needs of emerging, diverse biological products. In this study, a series of electrospun cellulose adsorbents were fabricated by adjusting their manufacturing conditions. A range of fiber diameters (400 to 600 nm) was created by changing the electrospinning polymer solution. Additionally, a range of porosities (0.4 to 0.7 v/v) was achieved by varying the laminating pressures on the electrospun sheets. The adsorbents were functionalized with different degrees of quaternary amine ligand density to create 18 prototype anion exchangers. Their morphology was characterized by BET nitrogen adsorption surface area, X-ray computed tomography, capillary flow porometry and scanning electron microscopy measurements. The physical characteristics of the adsorbents were used in an adapted semi-empirical model and compared to measured permeability data. Permeabilities of prototypes ranged from 10 -2 to 10 -4 mDarcy. The measured data showed good adherence to modelled data with possible improvements in acquiring wet adsorbent characteristics instead of dried material. Finally, the electrospun adsorbents were characterized for their binding capacity of model proteins of different sizes (diameters of 3.5 nm and 8.9 nm) and plasmid DNA. Static binding capacities ranged from 5 mg/ml to 25 mg/ml for the proteins and plasmid DNA and showed <20 % deviation from monolayer coverage based on BET surface area. Therefore, it was concluded that the electrospun adsorbents most likely adsorb monolayers of proteins and plasmid DNA on the surface with minimal steric hindrance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gyorgy Ovari reports financial support and equipment, drugs, or supplies were provided by Cytiva Stevenage. Thomas F. Johnson reports financial support was provided by Medical Research Council (MRC) Innovation Scholarship Grant. Thomas F. Johnson reports equipment, drugs, or supplies was provided by National Research Facility for Lab X-ray CT (NXCT). Gunnar Malmquist reports a relationship with Cytiva Sweden AB that includes: consulting or advisory and employment. Matthew Townsend reports a relationship with Cytiva that includes: employment. Farzad Foroutan reports a relationship with Cytiva that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Obtaining reliable and informative DNA data from soil samples is challenging due to the presence of interfering substances and typically low DNA yields. In this work, we prepared poly(ethylene glycol)-modified magnetic particles (PEG@Fe 3 O 4 ) for DNA purification. The particles leverage the facilitative effect of calcium ions (Ca 2+ ), which act as bridges between DNA and PEG@Fe 3 O 4 by coordinating with the phosphate groups of DNA and the hydroxyl groups on the particles. The addition of 2-propanol further enhances this Ca 2+ -mediated DNA adsorption by inducing a conformational change from the B-form to the more compact A-form of DNA. PEG@Fe 3 O 4 demonstrates a DNA adsorption capacity of 144.6 mg g -1 . When applied to the extraction of genomic DNA from soil samples, PEG@Fe 3 O 4 outperforms commercial kits and traditional phenol-chloroform extraction methods in terms of DNA yield and purity. Furthermore, we developed a 16-channel automated DNA extraction device to streamline the process and reduce the extraction time. The successful amplification of target bacterial and fungal amplicons underscores the potential of this automated, device-assisted method for studying soil microbial diversity.

The success of DNA analytical methods, including long-read sequencing, depends on the availability of high-quality, purified DNA. Previously, we developed a method and device for isolating high-molecular-weight (HMW) DNA for long-read sequencing using a high-salt gel electroelution trap. Here, we present an improved version of this method for purifying nucleic acids with high yield and purity from even the most challenging biological samples. The proposed method is a significant improvement over the previously published procedure, offering a simple, fast, and efficient solution for isolating HMW DNA and smaller DNA and RNA molecules. The method utilizes vertical gel electrophoresis in two nested, partially overlapping electrophoretic columns. The upper, smaller-diameter column has a thin layer of agarose gel at the bottom, which separates nucleic acids from impurities, and an electrophoresis buffer on top. After the target nucleic acid has been gel-purified on the upper column, a larger-diameter column with a layer of high-salt gel overlaid with electrophoresis buffer is inserted from below. The purified nucleic acid is then electroeluted into the buffer-filled gap between the separating gel and the high-salt gel, where excess counterions from the high-salt gel slow its migration and cause it to accumulate. The proposed vertical purification system outperforms the previously described horizontal system in terms of ease of use, speed, scalability, and compatibility with high-throughput workflows. Furthermore, the vertical system allows for the sequential purification of several nucleic acid species from the same sample using interchangeable salt-gel columns.

Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples., (© 2024. Springer Nature Limited.)

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis., (© 2024 Cold Spring Harbor Laboratory Press.)

As blood-feeding insects that feed on human hosts, bed bugs could be used in forensic investigations if they are present at a crime scene with no apparent evidence. This study describes how tropical bed bugs (Cimex hemipterus) can be used as forensic tools to collect valid human DNA samples. Short Tandem Repeat (STR) analysis was performed on collected bed bug samples, whereby the results indicate that the obtained quantities of human DNA are mostly substantial to facilitate a comprehensive genetic profiling process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Improvised explosive devices (IEDs) can be assembled directly from daily items and are easily purchasable and distributable internationally, owing to the absence of government export permits. Hence, their origins are not readily revealed, and they can pose significant adverse effects despite their low manufacturing costs. In this study, the feasibility of identifying fingerprints and deoxyribo nucleic acid (DNA) profiles in various IEDs and samples is investigated. Additionally, the relative positions of debris are identified to set the scope of on-site inspection at terrorist scenes. All samples are categorized into porous and non-porous materials, and LMG test, extraction, quantification, and short tandem repeat (STR) analysis are conducted to view the DNA profile. For fingerprinting, 1,2-IND and CA are utilized for development, followed by quality-control analysis. Although sample acquisition is impossible in some experiments, DNA profiling and fingerprint analysis are possible for all, thus allowing mapping to be performed. This study shows that even when terrorist bombing occurs, if evidence with minimal damage is detected at the scene, then STR profiles and fingerprints can be obtained at a level suitable for AFIS usage. Furthermore, accumulating mapping results from numerous experiments significantly aids in determining the scope of evidence acquisition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Wild animals, as a vital component of our natural world, serve a crucial role in preserving ecological equilibrium and biodiversity. By delving into the genetic constitution of wild animal populations, the evolutionary history, genetic diversity, and adaptation mechanisms could be explored, thereby informing conservation strategies and safeguarding the future of these species. In order to study the genetic information of wild animals, it is necessary to extract high purity and high concentration of wild animal genomic DNA. In this work, a hydrophobic magnetic deep eutectic solvent (HMDES) based vortexed extraction was developed for the extraction of genomic DNA from leopard cat (Prionailurus bengalensis), hairy-crowned deer (Elaphodus cephalophus) and muntjac (Muntiacus reevesi) muscle tissue, respectively. Extraction conditions like the pH value, extraction time, temperature and the amount of HMDES used were optimized by single-factor experiments. Under the optimized condition, genomic DNA could be selectively extracted from the three animal tissues. The limits of detection (LOD) and limits of quantification (LOQ) of the proposed method were 2.86 ng/μL and 8.66 ng/μL, respectively. Meanwhile, the relative standard deviation (RSD) of the method precision and repeatability were 1.64 % and 5.57 % at 20 ng/μL, showing the method has good precision and repeatability. After extraction, the DNA extracted into the HMDES droplets can be quickly recovered and the HMDES can be recycled and reused. The method proposed is a fast, environmental-friendly and high efficient extraction strategy for purification and enrichment of genomic DNA from leopard cat, hairy-crowned deer and muntjac tissues., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Innovative chiral capillary silica monoliths (CSMs) were developed based on DNA nanoflowers (DNFs). Baseline separation of enantiomers such as atenolol, tyrosine, histidine, and nefopam was achieved by using DNF-modified CSMs, and the obtained resolution value was higher than 1.78. To further explore the effect of DNFs on enantioseparation, different types of chiral columns including DNA strand containing the complementary sequence of the template (DCT)-modified CSMs, DNF 2 -modified CSMs, and DNF 3 -modified CSMs were prepared as well. It was observed that DNF-modified CSMs displayed better chiral separation ability compared with DCT-based columns. The intra-day and inter-day repeatability of model analytes' retention time and resolution kept desirable relative standard deviation values of less than 8.28%. DNF 2 /DNF 3 -modified CSMs were able to achieve baseline separation of atenolol, propranolol, 2'-deoxyadenosine, and nefopam enantiomers. Molecular docking simulations were performed to investigate enantioselectivity mechanisms of DNA sequences for enantiomers. To indicate the successful construction of DNFs and DNF-modified CSMs, various charaterization approaches including scanning electron microscopy, agarose gel electrophoresis, dynamic light scattering analysis, electroosmotic flow, and Fourier-transform infrared spectroscopy were utilized. Moreover, the enantioseparation performance of DNF-modified CSMs was characterized in terms of sample volume, applied voltage, and buffer concentration. This work paves the way to applying DNF-based capillary electrochromatography microsystems for chiral separation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

DNA typing of latent fingerprints is highly desirable to increase chances of individualization. We recovered DNA from Cyanoacrylate (CA) fumed fingerprints and used both GlobalFiler™ and ForenSeq™ DNA Signature Prep kits for DNA typing. For GlobalFiler™, samples were processed using a protocol modified for Low Template (LT)-DNA samples (half-volume reactions, 30 cycles) while for ForenSeq™ DNA Signature Prep, samples were processed using a standard protocol and fluorometer-based library quantitation. We evaluated genotyping success and quality of profiles in terms of completeness, Peak Height Ratio/Allele Coverage Ratio, presence of PCR artifacts and drop-in alleles. With GlobalFiler™, average autosomal STR (aSTR) profile completeness was 44.4% with 2-20 pg, 54.3% with 22-60 pg, and 95% with 64-250 pg DNA input. CODIS uploadable profiles were obtained in 2/10, 3/11, and 11/12 samples in these ranges. With ForenSeq™ DNA Signature Prep, average aSTR profile completeness was 19.7% with 1-20 pg and 45.2% with 22-47 pg but increased to 78.3% with 68-122 pg and 86.7% with 618-1000 pg DNA input. Uploadable profiles were obtained in 0/12, 4/11, 4/7, and 3/3 samples for these ranges. Results show very high sensitivity using both kits. Half-volume reactions and 30 cycles had minimal negative effect on Globalfiler™ profile quality, providing support for wider use after validation experiments to routinely improve results from LT samples. A standard protocol for the ForenSeq™ DNA Signature Prep kit was also highly successful with LT DNA obtained from CA-fumed fingerprints with additional information from isometric STR alleles and other markers., (© 2024 American Academy of Forensic Sciences.)

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole-genome single-nucleotide polymorphism (SNP) quality and quantity using next-generation sequencing (NGS). We present whole-genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double-stranded DNA libraries then enriched for whole-genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed-effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole-genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification., (© 2024 The Authors. Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

Oviductus Ranae is the dried oviduct from Rana dybowskii, a forest frog species with medicinal, tonic, and cosmetic properties. Due to the high price and resource shortage, counterfeit varieties of Oviductus Ranae often appear in the market. However, traditional identification methods cannot accurately differentiate between Oviductus Ranae and its adulterants. In this study, a rapid molecular identification method has been established. The method involves extracting genomic DNA in just 30 s using filter paper purification, species-specific rapid polymerase chain reaction (PCR) amplification, and finally, fluorescence detection of the products. It can accurately identify Oviductus Ranae and its three common adulterants in about 30 min, making the process simple, fast, and highly specific., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD 4 L did not., (© 2024. The Author(s).)

In 2019, the Texas Department of Public Safety (TXDPS) Texas Ranger Division (TRD) identified approximately 3300 registered sex offenders (RSOs) from whom a "lawfully owed" DNA sample was missing from the Federal Bureau of Investigation's Combined DNA Index System (CODIS). Lawfully owed DNA (LODNA) is defined as a DNA sample from a qualifying offender who should have had their sample entered into CODIS, but for unknown reasons did not. As a result of those findings, TXDPS then applied for and was awarded a grant from the Bureau of Justice Assistance's Sexual Assault Kit Initiative to collect DNA specimens from these RSOs, and to perform a statewide LODNA census. TXDPS TRD sought to determine: Are the missed DNA collection problems limited to RSO's or are they occurring among individuals with a qualifying arrest or conviction as specified by state law too? What processes are used to identify individuals who are eligible for DNA sample collection? How is an individuals' DNA collection eligibility conveyed to external agencies? The findings from TXDPS' LODNA census, identified 43,245 individuals who were likely eligible for DNA collection between 1995 and 2020, therefore indicating statewide DNA collection issues. Over 4 years, collection efforts pertaining to the aforementioned lawfully owed census, have yielded 5183 LODNA sample collections, and 276 CODIS hits. This manuscript aims to create an awareness within other agencies of the importance of implementing best practices to ensure the collection and upload of LODNA from every eligible individual., (© 2024 The Author(s). Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

The success of polymerase chain reaction (PCR) depends on the quality of deoxyribonucleic acid (DNA) templates. This study developed a cost-effective and eco-friendly DNA extraction system utilizing poly(3,4-dihydroxyphenylalanine)-modified cellulose paper (polyDOPA@paper). PolyDOPA@paper was prepared by oxidatively self-polymerizing DOPA under weak alkaline conditions and utilizing the adhesive property of polyDOPA on different materials. Compared to the uncoated cellulose paper, polyDOPA coating significantly enhances DNA adsorption owing to its abundant amino, carboxyl, and hydroxyl moieties. The DNA extraction mechanism using polyDOPA@paper was discussed. The maximum adsorption capacity of polyDOPA@paper for DNA was 20.7 μg cm -2 . Moreover, an automated extraction system was designed and fabricated using 3D printing technology. The device simplifies the operation and ensures the reproducibility and consistency of the results. More importantly, it eliminates the need for specialized training of operators. The feasibility of the polyDOPA@paper-based automated extraction system was evaluated by quantitatively detecting Escherichia coli in spiked milk samples via a real-time PCR. The detection limit was 10 2 cfu mL -1 . The results suggest that the system would have significant potential in detecting pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria , Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Assessments of biodiversity and ecosystem status can benefit from DNA metabarcoding as a means to streamline sample processing and specimen identification. Moreover, processing the fixation medium instead of the precious material introduces straightforward protocols that allow subsequent focus on certain organisms detected among the preserved specimens. In this study, we present a proof of concept via the analysis of freshwater invertebrate samples from the Tatra Mountain lakes (Slovakia). Besides highlighting a match between the lake-specific environmental conditions and the results of our fixative DNA metabarcoding, we observed an option to fine-tune the fixation time: to prefer two weeks over a day or a month. This effect emerged from the presence/absence of individual taxa rather than from coarse per-sample records of taxonomic richness, demonstrating that metabarcoding studies-and efforts to optimize their protocols-can use the robust metrics to explore even subtle trends. We also provide evidence that fixative DNA might better capture large freshwater species than terrestrial or meiofauna., (© 2024. The Author(s).)

Obtaining high-quality DNA suitable for long-read sequencing can be difficult for many types of tissues and cells, and it is a key step in current genomic studies. The challenge is even greater when it comes to isolating genomic DNA from mammalian spermatozoa, as DNA is tightly packed into a cell with a robust membrane rich in disulfide bonds. Here we describe a method for isolating high molecular weight DNA from Bovine commercial semen straws. This protocol includes a cleaning step to remove diluents and preservatives used for the long-term storage of the semen, which may affect long read sequencing. It is based on a simple salting-out method and avoid the use of spin columns, strong mixing or intensive centrifugation, in order to limit DNA fragmentation. However, we have adapted this protocol to facilitate the disruption of cell membranes and disulfide bonds with strong chaotropic and reducing agents. The average size of the fragments produced was approximately 49 kb, ranging from 25 to 85 kb, according to the femto pulse profiles.This method was used to isolate DNA from semen straws, more than 80 of them were successfully sequenced using the Continuous Long-Read (CLR) sequencing mode on the PacBio SequelII platform to study genome diversity and notably to detect large structural variations within genomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Denis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA retrieval methods traditionally used during forensic evidence recovery including swabbing and tape lifting, can have limited effectiveness when used on porous, rough substrates such as bricks and carpet. This is possibly due to the DNA material being dispersed and unreachable for surface sampling techniques. In this evaluation we investigated the effectiveness of the Microbial Wet-Vacuum System (M-Vac®; M-Vac® Systems, Inc., Sandy, UT), as it has been reported to retrieve greater amounts of DNA material from challenging exhibits. A four-stage evaluation was conducted, starting with seeding carpet and brick substrates with a known donor's saliva in two dilutions and comparing the DNA recovery of tape lifting, swabbing, and the M-Vac®. A victim struggle scenario on carpet was then mimicked to compare trace DNA recovery by each method. Two mock scenarios were also conducted; a shirt was submerged in a creek bed for a period of five days to sample for the wearer's DNA, and a car boot was sampled to assess the possibility of recovering a victim's DNA amongst background DNA from the usual car occupants. Finally, the compatibility of the M-Vac® sampling process was optimised for the fully automated DNA lysis and extraction platforms used in the NSW (Australia) jurisdiction by comparing filter subsampling methods. The results from the study were mixed. For bricks, none of the collection methods were effective in retrieving DNA. On carpet, the M-Vac® retrieved the greatest quantities of DNA from the saliva-seeded samples, however, tape lifts outperformed all methods for 'touch' DNA recovery. The M-Vac® retrieved the greatest amount of DNA from the t-shirt recovered from a creek bed as it was able to retrieve the embedded DNA. The final mock case car boot scenario resulted in greater victim DNA recovery from tape lifts, with the M-Vac® more likely to recover mixtures too weak and/or complex to be interpreted. Finally, operational considerations regarding the compatibility of the M-Vac® system with fully automated DNA lysis and extraction are discussed. Considering the substantial time and cost to deploy the M-Vac®, it is recommended to be utilised in casework only after swabbing and tape lifting methods have failed to yield sufficient DNA material, where the substrate properties would likely benefit from the M-Vac's® niche capabilities for retrieving embedded DNA, and low levels of background DNA may be anticipated., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The work describes the use of SYBR Gold to improve the detection sensitivity of plasmid DNA topoisomers by capillary gel electrophoresis with laser induced fluorescence in an uncoated capillary. The impact of different dyes, including ethidium bromide, SYBR Green and SYBR Gold, was compared based on detection and separation of DNA plasmid topoisomers. Use of SYBR Gold enabled improvement of detection sensitivity by 15-fold while maintaining good separation resolution of the different topoisomers. The baseline dropped with the use SYBR Gold but was overcome by the employment of a capillary with longer ineffective length (40 vs. 20 cm). Separation resolution and reproducibility were impacted by the concentration of SYBR Gold and hydroxypropyl methylcellulose. With the use of a short capillary (10 cm effective length and 50 cm total length), fast separations of supercoiled, linear, open circular, and other isoforms were accomplished within 8 min. Appropriate capillary cleaning with 0.1 M sodium hydroxide/0.1 M hydrochloric acid and capillary storage with 0.1 M hydrochloric acid ensured good separation reproducibility of 217 runs during an extended period of use., (© 2024 Wiley‐VCH GmbH.)

High-quality DNA is an important guarantee to start downstream experiments in many biological and medical research areas. Magnetic particle-based DNA extraction methods from blood mainly depend on electrostatic adsorption in a low-pH environment. However, the strong acidic environment can influence the DNA stability. Herein, a polydopamine-functionalized magnetic particle (PDA@Fe 3 O 4 )-based protocol was developed for DNA extraction from whole blood samples. In the protocol, Mg 2+ and Ca 2+ were utilized to bridge the adsorption of DNA by PDA@Fe 3 O 4 via the metal-mediated coordination. Isopropanol was found to efficiently promote DNA adsorption by triggering the change of the conformation of DNA from B-form to more compact A-form. In 50 % isopropanol solution, the DNA adsorption efficiency was nearly 100 % in the presence of 0.5 mM Ca 2+ or 1.5 mM Mg 2+ . The role of metal ions and isopropanol in DNA adsorption was explored. The protocol averts the strong acidic environment and PCR inhibitors, such as high concentrations of salt or polyethylene glycol. It demonstrates superiority in DNA yield (59.13 ± 3.63 ng μL -1 ) over the commercial kit (27.33 ± 4.98 ng μL -1 ) and phenol-chloroform methods (37.90 ± 0.47 ng μL -1 ). In addition, to simplify the operastion, an automated nucleic acid extraction device was designed and fabricated to extract whole genomic DNA from blood. The feasibility of the device was verified by extracting DNA from cattle and pig blood samples. The extracted DNA was successfully applied to discriminate the beef authenticity by a duplex PCR system. The results demonstrate that the DNA extraction protocol and the automated device have great potential in blood samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Adulteration of meat is a global issue, necessitating rapid, inexpensive, and simple on-site testing methods. Therefore, the present study aimed to develop a one-minute toothpick-based DNA extraction method, a handheld microfluidic chip, and a smartphone-controlled portable analyzer for detecting multiple meat adulterations. A toothpick was inserted into the meat to promote DNA release and adsorption. Furthermore, a handheld microfluidic chip was designed for DNA elution on toothpicks and fluid distribution. Finally, a smartphone-actuated portable analyzer was developed to function as a heater, signal detector, and result reader. The portable device comprises a microcontroller, a fluorescence detection module, a step scanning unit, and a heating module. The proposed device is portable, and the app is user-friendly. This simple design, easy operation, and fast-response system could rapidly detect as little as 1% of simulated adulterated samples (following UK standards) within 40 min at a cost of less than USD 1 per test., Competing Interests: Declaration of competing interest The authors declare no competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Slalom chromatography (SC) re-emerged in 2024 due to the availability of low adsorption ultra-high pressure liquid chromatography (UHPLC) packed columns/instruments and large modalities being investigated in the context of cell and gene therapies. The physico-chemical principles of SC retention combined with hydrodynamic chromatography (HDC) exclusion have been recently reported. In SC, DNA macromolecules are retarded because: (1) they can be stretched to lengths comparable to the particle diameter, and (2) their elastic relaxation time is long enough to maintain them in non-equilibrium extended conformations under regular UHPLC shear flow conditions. Here, a quantitative HDC-SC retention model is consolidated. A general plate height model accounting for the band broadening of long DNA biopolymers along packed beds is also derived for supporting method development and predicting speed-resolution performance in SC. For illustration, the chromatographic speed-resolution properties in SC are predicted for the separation of specific critical pairs (4.0/4.5, 10/11, and 25/27 kbp) of linear dsDNA polymers. The calculations are performed for two available custom-made particle sizes, d p = 1.7 and 2.5μm, at a constant pressure of 10,000 psi. The predictions are directly validated from experimental data acquired using low adsorption MaxPeak TM 4.6 mm i.d. Columns packed with 1.7μm BEH TM 45 Å (15 cm long column) and 2.5μm BEH 125 Å (30 cm long column) Particles, and by injecting six linear dsDNAs (λ DNA-Hind III Digest). The LC system is very low dispersion ACQUITY TM UPLC TM I-class PLUS System, and the mobile phase is a 100 mM phosphate buffer at pH 8. Maximum resolution is always achieved when the average extended lengths of linear dsDNAs are equal to a critical length, which is proportional to the particle diameter and to the square root of the applied shear rate. Most advantageously, the experimental results reveal that the relaxation times of linear dsDNAs observed under shear flow conditions are two orders of magnitude shorter than those expected in the absence of flow: this enables the detection of the longest linear dsDNAs up to 25 kbp without irremediable loss in column performance. Finally, the retention-efficiency model elaborated in this work can be used to rapidly anticipate and develop methods (selection of particle size, column length, and operating pressure) for any targeted DNA and time-resolution constraints., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier B.V. All rights reserved.)

Most of the sexual assault casework samples are of mixed sources. Forensic DNA laboratories are always in the requirement of a precise technique for the efficient separation of sperm and non-sperm DNA from mixed samples. Since the introduction of the differential extraction technique in 1985, it has seen significant advancements in the form of either chemicals used or modification of incubation times. Several automated and semi-automated techniques have also adopted the fundamentals of conventional differential extraction techniques. However, lengthy incubation, several manual steps, and carryover over non-sperm material in sperm fraction are some of the major limitations of this technique. Advanced cell separation techniques have shown huge promise in separating sperm cells from a mixture based on their size, shape, composition, and membrane structure and antigens present on sperm membranes. Such advanced techniques such as DEParray, ADE, FACS, LCM, HOT and their respective pros and cons have been discussed in this article. As current-day forensic techniques should be as per the line of Olympic slogan i.e., faster, higher, stronger, the advanced cell separation techniques show a huge potential to be implemented in the casework samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The combination of multi-omic techniques, such as genomics, transcriptomics, proteomics, metabolomics and epigenomics, has revolutionised studies in medical research. These techniques are employed to support biomarker discovery, better understand molecular pathways and identify novel drug targets. Despite concerted efforts in integrating omic datasets, there is an absence of protocols that integrate all four biomolecules in a single extraction process. Here, we demonstrate for the first time a minimally destructive integrated protocol for the simultaneous extraction of artificially degraded DNA, proteins, lipids and metabolites from pig brain samples. We used an MTBE-based approach to separate lipids and metabolites, followed by subsequent isolation of DNA and proteins. We have validated this protocol against standalone extraction protocols and show comparable or higher yields of all four biomolecules. This integrated protocol is key to facilitating the preservation of irreplaceable samples while promoting downstream analyses and successful data integration by removing bias from univariate dataset noise and varied distribution characteristics., (© 2024. The Author(s).)

An approach for the controllable separation and concentration of nucleic acid using a circular nonuniform electric field was proposed and developed. Using six different lengths of DNA molecules as standard samples, the distribution of the gradient electric field was increased from the outer circular electrode to the inner rod-shaped electrode, contributing to the migration of DNA molecules at a velocity gradient towards the region with the strongest inner electric field. The DNA molecules were arranged in a distribution of concentric circles that aligned with the distribution of concentric equipotential lines. The concentration of DNA multiplied with the alternation of radius. As a result, this platform allowed simultaneous DNA separation, achieving a resolution range of 1.17-3.03 through an extended electrophoresis time, resulting in enhanced concentration factors of 1.08-6.27. Moreover, the manipulation of the relative height of the inner and outer electrodes enabled precise control over the distribution and the deflection degree of electric field lines, leading to accurate control over DNA deflection., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Efficient, mild, and reversible adsorption of nucleic acids onto nanomaterials represents a promising analytical approach for medical diagnosis. However, there is a scarcity of efficient and reversible nucleic acid adsorption nanomaterials. Additionally, the lack of comprehension of the molecular mechanisms governing their interactions poses significant challenges. These issues hinder the rational design and analytical applications of the nanomaterials. Herein, we propose an ultra-efficient nucleic acid affinity nanomaterial based on programmable lanthanide metal-organic frameworks (Ln-MOFs). Through experiments and density functional theory calculations, a rational design guideline for nucleic acid affinity of Ln-MOF was proposed, and a modular and flexible preparation scheme was provided. Then, Er-TPA (terephthalic acid) MOF emerged as the optimal candidate due to its pore size-independent adsorption and desorption capabilities for nucleic acids, enabling ultra-efficient adsorption (about 150% mass ratio) within 1 min. Furthermore, we elucidate the molecular-level mechanisms underlying the Ln-MOF adsorption of single- and double-stranded DNA and G4 structures. The affinity nanomaterial based on Ln-MOF exhibits robust nucleic acid extraction capability (4-fold higher than commercial reagent kits) and enables mild and reversible CRISPR/Cas9 functional regulation. This method holds significant promise for broad application in DNA/RNA liquid biopsy and gene editing, facilitating breakthroughs in analytical chemistry, pharmacy, and medical research.

The generation of high-quality DNA profiles from trace amounts of DNA continues to be an issue in forensic casework. Several methods have been proposed over the years to increase recovery rates for low input DNA, including purification of PCR products, an increase in PCR cycle numbers and increasing injection time or voltage during electrophoresis. In this study, the characteristics of DNA profiles generated using QIAGEN MinElute® purification of Promega PowerPlex® 21 amplified products for low DNA input samples, ranging from 80 pg down to 4 pg, were evaluated. MinElute® purification was found to be a simple, effective and time efficient method, which can greatly improve the resolution of amplified PCR products, recovering 100% of donor concordant alleles from as little 16 pg of input template DNA and generating sufficient allelic information for interpretation from as low as 4 pg inputs. However, as is commonly observed with low template DNA samples, the results exhibited extensive disparity in the effects of stochastic variation in amplification, including increased heterozygote peak height imbalance, stutter ratios and instances of allelic drop-in and drop-out, both within and between replicates. As such, it is important that the extent and variability of these stochastic effects are appropriately incorporated in the development of robust profile interpretation guidelines for DNA profiles generated from purified PCR products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Weak and partial DNA profiles are commonly encountered within forensic casework due to amplification of low DNA input samples. One option for increasing allelic detection in such samples is the purification of amplified PCR product using commercially available column-based methods. In this study, four commercially available post-PCR purification methods, QIAGEN MinElute®, Independent Forensics Amplicon™ Rx, Millipore Microcon® and Thermo Fisher Scientific ExoSAP-IT™ were evaluated, comparing the quality of PowerPlex® 21 DNA profiles produced to the standard DNA profile generated prior to purification. An increased detection of alleles above the analytical threshold was observed following purification with the MinElute®, Amplicon™ Rx and Microcon® methods, allowing informative DNA profiles to be recovered using as little as 8 pg DNA. However, post-PCR purification using the ExoSAP-IT™ kit was unsuccessful, with no alleles detected above analytical threshold in samples with ≤16 pg DNA. The MinElute® kit was selected for optimisation on the basis of DNA profile quality, including increased detection of alleles and minimal artefacts. The MinElute® method was optimised by evaluating the number of washes and final elution buffer volume, resulting in a further increase in detection of alleles by reducing the elution buffer volume. Overall, this study showed that PowerPlex® 21 DNA profiles from low input DNA can be successfully enhanced by employing the MinElute® post-PCR purification method., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70-80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone. The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DNA mixture deconvolution in the forensic DNA community has been addressed in a variety of ways. "Front-end" methods that separate the cellular components of mixtures can provide a significant benefit over computational methods as there is no need to rely on models with inherent uncertainty to generate conclusions. Historically, cell separation methods have been investigated but have been largely ineffective due to high cost, unreliability, and the lack of proper instrumentation. However, the last decade has given rise to more innovative technology that can target and recover cells more effectively. This study focuses on the development and optimization of a method to selectively label and recover male cells in a mixture of male and female epithelial cells using a Y-chromosome labeling kit with DEPArray™ technology, whereby male cells are labeled and recovered into a single extraction-ready tube. Labeling efficiency was tested using freshly collected and aged buccal swabs where 70%-75% and 38% of male cells were labeled, respectively, with less than 1% false positives. DEPArray™ detection was assessed using single buccal epithelial cells where approximately 80% of labeled cells were identified as male. Mixtures (1:1, 1:10, male to female) yielded profiles that were predominantly single source male or those in which the male component was more easily interpreted. The male-specific labeling method was demonstrated to be both robust and reliable when used on freshly collected cells. While the DEPArray™ meditated detection and recovery had notable limitations, it still improved the interpretation of the male component in same-cell mixtures in more recently collected samples., (© 2024 The Authors. Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

Research into the recovery of DNA from illicit drug samples has shown it is possible to get forensically useful profiles from such substrates. However, it is not yet known if the different physical states that drugs can be found in influences the quantity and quality of DNA that can be recovered or what is the best sampling method to adopt for powdered samples. This research used acetaminophen in four different states - large crystalline, powder, in solution, or residue - to determine the efficacy of current DNA technology in recovery and analysis of the resulting sample. Five replicates of each were prepared. Human blood was deposited on or mixed with the drug and left for 1 hour. The surface of the drug was sampled by wet/dry swabbing (where appropriate), or the entire sample was deposited in a tube, and the DNA then extracted using DNA-IQ™. The amount of DNA recovered (ng), degradation index, number of PCR cycles (Ct) required for the IPC to reach threshold, number of alleles in the DNA profile and average peak height (APH) were assessed. All samples, irrespective of the physical state they were collected from, returned full DNA profiles that corresponded to the DNA profile of the blood donor, with no degradation or inhibition detected. It was also found the wet/dry swabbing method returned higher levels of DNA than inclusion of the entire sample into the tube for powdered acetaminophen and the appropriate method to use will be dependent on casework circumstances. The findings of this research further develops our understanding of the recovery of DNA from drugs, and supports the need for further investigation to understand under what conditions DNA can be recovered from illicit substances., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal., Methods: Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability., Results: We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices., Conclusions: This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing., (© Association for Diagnostics & Laboratory Medicine 2024.)

Telomere Length (TL) and integrity is significantly associated with age-related disease, multiple genetic and environmental factors. We observe mouse genomic DNA (gDNA) isolation methods to have a significant impact on average TL estimates. The canonical qPCR method does not measure TL directly but via the ratio of telomere repeats to a single copy gene (SCG) generating a T/S ratio. We use a monochromatic-multiplex-qPCR (mmqPCR) method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction. We demonstrate that TL measurements, from murine gDNA, isolated via Spin Columns (SC) and Magnetic Beads (MB), generate significantly smaller T/S ratios compared to gDNA isolated via traditional phenol/chloroform methods. The former methods may impede correct TL estimation by producing non representative fragment sets and reducing qPCR efficacy. This work highlights discrepancies in TL measurements due to different extraction techniques. We recommend the use of gDNA isolation methods that are shown to preserve DNA length and integrity, such as phenol/chloroform isolation. We propose that widely used high throughput DNA isolation methodologies can create spurious associations within a sample set, thus creating misleading data. We suggest that published TL associations should be revisited in the light of these data.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fish species are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals to permit identification of their genotype. The current standard procedure to sample DNA is fin clipping, which involves anaesthetising individuals and removing a portion of the caudal fin. While fin clipping reliably generates good quality DNA samples for downstream applications, there is evidence that it can alter health and welfare, and impact the fish's behaviour. This in turn can result in greater variation in the data collected. In a recent study we adapted a skin swabbing protocol to collect DNA from small-bodied fish, including sticklebacks and zebrafish, without the use of analgesics, anaesthetics or sharp instruments. A rayon-tipped swab was used to collect mucus from the flank of the fish, which was then used for DNA extraction. We subsequently demonstrated that compared to fin clipping, skin swabbing triggered fewer changes in stress axis activation and behaviour. We also found that gene expression and behaviour data collected from swabbed fish were less variable than similar data collected from fish that had been fin clipped. This potentially allows smaller sample sizes in experimental groups to be used after skin swabbing, thereby reducing animal use. Here we provide a detailed protocol explaining how to collect DNA samples from small laboratory fish using skin swabs., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Tilley C et al.)

We recently identified a class of small cytosolic double-stranded DNA (scDNA) approximately 20-40 bp in size in human and mouse cells. Here, we present a protocol for scDNA isolation from cultured murine cells. We describe steps for cytosolic compartment separation, DNA isolation in the cytosolic fraction using phenol-chloroform extraction, and ethanol precipitation. We then detail procedures for denaturing purified cytosolic DNA through urea polyacrylamide gel electrophoresis and obtaining scDNA in the cytosolic DNA fraction via gel purification. For complete details on the use and execution of this protocol, please refer to Liu et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.

The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies., (© 2024. The Author(s).)

DNA quality is of paramount importance for molecular biology research. This study aimed to assess the DNA extracted from residual blood clots after serological testing, focusing on the impact of blood clot segments, extraction kits, temporary storage durations (TSDs), and thawing methods on DNA quality. We divided the residual blood clot column (BCC) from healthy donors into three segments and utilized two different extraction kits. The BCCs were subjected to four TSDs at 4°C (7 days, 10 days, 1 month, and 2 months) and three thawing methods (4°C, room temperature, and 37°C). We found that the TIANamp Blood Clot DNA Kit yielded consistently high-quality DNA from each segment with stable A260/280 and A260/230 ratios. The DNA yield showed a strong positive correlation with leukocyte concentration, and a satisfactory median DNA yield of 28.79 μg/g BCC was obtained across all segments. DNA integrity, as measured by the DNA integrity number and DNA fragment peak size, decreased with increasing TSD at 4°C, with a notable decrease after 10 days of storage. Thawing at 37°C resulted in the lowest DNA fragment peak size. In conclusion, BCC could be an ideal DNA source with satisfactory yield and purity. A prolonged TSD at 4°C leads to an obvious decrease in DNA integrity, and thawing the frozen BCC at 37°C decreases DNA fragment sizes. To maintain DNA integrity, BCCs should be cryopreserved as soon as possible after short TSDs at 4°C and thawed at 4°C., (© 2024 John Wiley & Sons Ltd.)

Nucleic acid therapeutics have the potential to revolutionize the biopharmaceutical industry, providing highly effective vaccines and novel treatments for cancers and genetic disorders. The successful commercialization of these therapeutics will require development of manufacturing strategies specifically tailored to the purification of nucleic acids. Membrane technologies already play a critical role in the downstream processing of nucleic acid therapeutics, ranging from clarification to concentration to selective purification. This review provides an overview of how membrane systems are currently used for nucleic acid purification, while highlighting areas of future need and opportunity, including adoption of membranes in continuous bioprocessing., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The main aim of this work was to validate the detection of saliva samples from denim, cotton, and polyester fabrics aged up to six months, applying rapid immunochromatographic tests resulting in the analysis of nuclear and mitochondrial DNA recovered. A comparison was also carried out between two saliva detection tests, Laboratory and Crime Scene. 50 μl saliva samples (three per time and test) were deposited on denim, cotton, and polyester fabrics. After 1, 3, 7, 14, 21, 30, 60, 90, 150, and 180 days of storage at room temperature, the samples were recovered by swabbing and detected by SERATEC ® Amylase (Laboratory) test and SERATEC ® SALIVA CS (Crime Scene) test (SERATEC ® , Göttingen, Germany). DNA was isolated from the swab extraction buffer applying a silica-based methodology, and quantified based on fluorescent and human-specific quantifications. Then, it was submitted to STR profiling and mtDNA sequencing. According to our results, saliva stains up to six months after deposition remain valid specimens. The intensity of the bands varied among fabric type and time. Total DNA was successfully recovered from all tested samples, though with the limitations of obtaining partial nuclear DNA profiles from the oldest samples. In contrast, complete characterization of mtDNA was achieved from all samples. Lab and CS tests performed similar on the detection of saliva, as well as, DNA yield and profiling. Future research will be able to expand these results, analyzing the stability of other body fluids and the sensitivity of rapid immunochromatographic tests to detect them., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na 2 EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell ® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index ( p -values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Background: Diagnosing imprinting defects in neonates and young children presents challenges, often necessitating molecular analysis for a conclusive diagnosis. The isolation of genetic material from oral swabs becomes crucial, especially in settings where blood sample collection is impractical or for vulnerable populations like newborns, who possess limited blood volumes and are often too fragile for invasive procedures. Oral swab samples emerge as an excellent source of DNA, effectively overcoming obstacles associated with rare diseases., Methods: In our study, we specifically addressed the determination of the quality and quantity of DNA extracted from oral swab samples using NaCl procedures., Results: We compared these results with extractions performed using a commercial kit. Subsequently, the obtained material underwent MS-HRM analysis for loci associated with imprinting diseases such as Prader-Willi and Angelman syndromes., Conclusions: Our study emphasizes the significance of oral swab samples as a reliable source for obtaining DNA for MS-HRM analysis. NaCl extraction stands out as a practical and cost-effective method for genetic studies, contributing to a molecular diagnosis that proves particularly beneficial for patients facing delays in characterization, ultimately influencing their treatment.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization., Competing Interests: The authors declare that they have no competing interests., (© 2024 Dabrowski et al.)

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

High-cost DNA extraction procedures pose significant challenges for budget-constrained laboratories. To address this, we introduce OpenCell, an economical, open-source, 3-in-1 laboratory device that combines the functionalities of a bead homogenizer, a microcentrifuge, and a vortex mixer. OpenCell utilizes modular attachments that magnetically connect to a central rotating brushless motor. This motor couples to an epicyclic gearing mechanism, enabling efficient bead homogenization, vortex mixing, and centrifugation within one compact unit. OpenCell's design incorporates multiple redundant safety features, ensuring both the device's and operator's safety. Additional features such as RPM measurement, programmable timers, battery operation, and optional speed control make OpenCell a reliable and reproducible laboratory instrument. In our study, OpenCell successfully isolated DNA from Spinacia oleracea (spinach), with an average yield of 2.3 μg and an A260/A280 ratio of 1.77, demonstrating its effectiveness for downstream applications such as Polymerase Chain Reaction (PCR) amplification. With its compact size (20 cm x 28 cm x 6.7 cm) and lightweight design (0.8 kg), comparable to the size and weight of a laptop, OpenCell is portable, making it an attractive component of a 'lab-in-a-backpack' for resource-constrained environments in low-and-middle-income countries and synthetic biology in remote field stations. Leveraging the accessibility of 3D printing and off-the-shelf components, OpenCell can be manufactured and assembled at a low unit cost of less than $50, providing an affordable alternative to expensive laboratory equipment costing over $4000. OpenCell aims to overcome the barriers to entry in synthetic biology research and contribute to the growing collection of frugal and open hardware., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)