DNA Extraction Process Research Articles

Comparative evaluation of DNA synthesis for qPCR analysis from oral squamous cell carcinoma tissues - A rapid and robust isolation technique for gene expression studies

BackgroundDNA isolation from biological materials is the initial step in several bioanalytical processes, including the polymerase chain reaction (PCR). This procedure works best with pure DNA devoid of potential amplification reaction inhibitors. Since the quantity and quality of biological samples are limited, it is essential to extract as much DNA as possible from the original sample. The solid-phase extraction technique is frequently used to purify DNA. In this process, the DNA is adsorbed onto a solid support, any contaminants from the reaction are eliminated by washing, and the purified DNA is then eluted from the support. The quality and concentration of DNA have a direct effect on the gene analysis procedure. Therefore, before interpreting the results of PCR-based diagnostic assays, it is imperative to confirm the DNA preparation and integrity, particularly if no pathogenic DNA is identified. MethodsA comparative study was carried out using two standardized protocols for DNA synthesis in comparison with a test protocol incorporating carrier RNA and proteinase K from a viral detection kit. The quality and quantity of the synthesized DNA are assessed by qPCR analysis for gene amplification of metastasis suppressor genes NDRG1, RhoGDI, and KISS 1. Results and conclusionThe test protocol showed higher yields of DNA in comparison to the standard protocol. The concentration of DNA obtained was validated by the concentration of gene expressed from q PCR analysis. The gene expression was significantly higher when the test protocol method was followed for DNA synthesis. Thus the study validates the potential of nucleic acid carrier RNA molecules to improve DNA extraction processes used in tissue samples for gene analysis procedures.

ADVANCING INDIVIDUAL FORAMINIFERA ANALYSIS BY COMBINING MOLECULAR, MORPHOMETRIC, AND TRACE ELEMENT GEOCHEMISTRY

ABSTRACT Planktic foraminifera are widely used as paleoclimate proxies. Foraminiferal species are identified morphologically, but research has revealed that many species contain cryptic genetic diversity. Here, we advance a workflow analyzing the genetics, morphology, and geochemistry from individual foraminifera specimens, using Globigerina bulloides as the test species. The impact of the DNA extraction process is assessed by comparing the trace element geochemistry and test morphometrics of DNA extracted vs. control specimens. Imaging revealed highly variable morphologies within the same genotype. Physical properties of the test were not influenced by extraction. DNA extraction did not impact Mg/Ca and B/Ca trace element ratios, common proxies for paleothermometry and carbonate chemistry, respectively. However, DNA extraction did slightly elevate some trace element ratios (Zn, Ba, and Sr) and suggests that additional cleaning may be required. This workflow provides a roadmap for obtaining genetic, morphometric, and geochemical data from the same specimens, and for elucidating cryptic diversity within species.

A super convenient and specific CRISPR/Cas12a diagnostic platform for toxigenic Burkholderia gladioli based on virulence genes

In recent years, food poisoning cases caused by toxigenic Burkholderia gladioli pv. cocovenenans (BGC) have been frequently reported in Asian countries like China and Indonesia, which seriously threaten food safety and public health. BGC is the only subspecies within the Burkholderia gladioli species that can cause food poisoning in humans, but there is a high degree of sequence homology between the different subspecies, making detection and differentiation difficult. In this study, a specific DNA target was screened out from the bongkrekic acid biosynthentic gene cluster. A rapid, sensitive, and highly specific CRISPR/Cas12a molecular diagnostic platform was developed for the first time to differentiate between toxigenic and non-toxigenic subspecies. Genomic DNA was extracted from the samples after a simple boiling process. The sensitivity was evaluated from three different levels, the genomic DNA sensitivity was determined to be 10−4 ng/μL, the target DNA sensitivity was determined to be 14.9 copies/μL, and the bacterial suspension sensitivity was confirmed to be 1.52 CFU/mL. The detection limit of BGC in artificially contaminated coconut water samples was determined to be 1.50 CFU/mL without any microbial enrichment. Including sample pretreatment and DNA extraction process, the whole detection of BGC in samples can be completed within 60 min under the assistance of some small and portable devices. This super convenient diagnostic platform is suitable for food field testing and provides a theoretical basis for the specific identification of BGC.

Enhancing Colorimetric Detection of Nucleic Acids on Nitrocellulose Membranes: Cutting-Edge Applications in Diagnostics and Forensics.

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology.

Microbial Diversity Assessment in Soil from Dumping Sites: Isolation, DNA Extraction and Electrophoretic Analysis

This study aims to investigate the microbial diversity present in soil samples collected from various dumping sites, which are known for their complex and heterogeneous waste environments. The primary objective is to isolate and characterize the microbial communities inhabiting these soils, providing insights into their composition and potential ecological roles. Soil samples were collected by digging 5 cm deep at multiple dumping locations to ensure a representative sampling of the microbial populations. The collected soil samples underwent a serial dilution process, extending up to 10^-9 and 10^-10 dilutions, to facilitate the isolation of individual microbial colonies. These diluted samples were then inoculated onto Standard Potato Dextrose Agar (PDA) media, a nutrient-rich medium conducive to microbial growth. Following an incubation period, distinct bacterial colonies were observed and isolated for further analysis. DNA was extracted from the isolated bacterial colonies using a standard DNA extraction protocol. The quality and purity of the extracted DNA were assessed through gel electrophoresis, a technique that separates DNA fragments based on their size. Remarkably, the electrophoretic analysis revealed clear and distinct DNA bands along with RNA bands, indicating successful extraction of high-quality microbial DNA. Notably, this was achieved without the use of proteinase treatment, which is typically employed to remove protein contaminants from DNA samples. The results of this study underscore the robustness of the methodologies employed in isolating and characterizing microbial DNA from soil samples collected at dumping sites. The presence of distinct DNA and RNA bands highlights the effectiveness of the DNA extraction process, even in the absence of proteinase treatment. These findings provide valuable insights into the microbial diversity within dumping site soils, suggesting a rich and varied microbial community that may play significant roles in soil health and waste decomposition.

Prevalence of Toxoplasma gondii Infections in Pregnant Women and Small Ruminants: A Molecular Diagnostic Approach

Background: Toxoplasma gondii is a globally prevalent parasite with significant health implications, particularly for pregnant women and immunocompromised individuals. Infections during pregnancy can result in serious outcomes such as miscarriage, stillbirth, or congenital disabilities. This study aimed to assess the prevalence of T. gondii infection among pregnant women and small ruminants in Al-Anbar province, Iraq, using serological and molecular diagnostic techniques. Methods: Blood samples were collected from 165 pregnant women and 100 small ruminants (sheep and goats) between January and May 2021. Serological screening was conducted using the Humatex-Toxo rapid test to detect antibodies against T. gondii. DNA was extracted from the blood samples, and nested polymerase chain reaction (PCR) targeting the T. gondii B1 gene was used for molecular detection. The infection rates were analyzed statistically using ANOVA and Chi-square tests, with significance set at p<0.05. Results: The serological test identified T. gondii antibodies in 32.7% of pregnant women, while the PCR detected a higher infection rate of 40%. Among the small ruminants, PCR revealed an overall infection rate of 10%, with sheep showing a higher rate (16%) compared to goats (4%). The results indicate that PCR is more sensitive than serological testing, particularly for identifying active infections. Additionally, the higher infection rate in young women may be linked to lifestyle, environmental exposure, and dietary habits, while seasonal factors likely play a role in infection distribution. Conclusion: Nested PCR is a more effective method for detecting T. gondii in pregnant women, offering a precise tool for identifying infections and potential causes of abortion. The lower infection rates in small ruminants suggest a reduced zoonotic risk in the study area, although continued monitoring is essential. Negative PCR results may be due to inhibitors in the DNA extraction process or the parasite’s localization in tissues rather than blood. The findings highlight the importance of molecular diagnostics in public health strategies aimed at reducing T. gondii infections in high-risk populations.

Optimization of DNA Extraction for ITS/U4U3 analysis of Rhipsalis baccifera

Genetic analysis of plants relies on high yields of pure DNA. For Rhipsalis baccifera this represents a great challenge since its plant tissue can accumulate large amounts of mucilage, polysaccharides, polyphenols and secondary metabolites, which co-purify with amplifiable DNA. These contaminating compounds lead to a poor yield and prevent acces to PCR-based analysis. These contaminating compounds lead to a poor DNA yield and prevent access to PCR-based analysis. A number of factors, including choice of plant tissue, tissue preparation, and modifications of the extraction buffer, can impact on DNA extraction process. In this study, four different DNA extraction procedures were tested aiming to develop a simple protocol based on CTAB buffer. The obtained results showed that the use of the outer cuticle of old lyophilized tissue allowed reliable results in R. baccifera plants with a good purity ranging from 1.6 to 1.8 and high DNA yield yield ? 500 ng ?L-1.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

Molecular survey of tick-borne infectious agents in cricetid rodents (Rodentia: Cricetidae) in Central and Southern Chile.

Tick-borne infectious agents (TBIAs) include several bacteria and protozoa that can infect vertebrates, including humans. Some of these agents can cause important diseases from both a public health perspective, such as Lyme disease, and from an animal health and production viewpoint, such as Texas fever. In Chile, several studies have assessed the presence of tick-borne disease agents in vectors and mammal hosts, mainly in the northern regions, but few studies have assessed the presence of these agents in Central and Southern Chile. This study aimed to assess the presence of three groups of TBIAs-Borrelia, Anaplasmataceae, and Piroplasmida-in cricetid rodents of Central and Southern Chile. A total of 207 specimens from 13 localities between the O'Higgins and Los Lagos regions were captured. DNA was extracted from the liver and spleen, and subsequently underwent polymerase chain reaction (PCR) amplification targeting the 16S rRNA, flaB, and 18S rRNA genes to detect DNA from Borrelia, Anaplasmataceae, and Piroplasmida, respectively. Although no DNA from these TBIAs was detected, the DNA extraction process was validated by optimal DNA purity ratios (an A260/A280 ratio within the 1.6-2.0 range) and successful internal control amplification in all samples. These results, in addition to findings from previous reports, suggest a very low prevalence of these TBIAs in the rodent population studied. Further research into the factors influencing the presence of these agents and their vectors will provide insight into the reasons underlying this low prevalence.

Proficiency testing for event-specific quantification of genetically modified maize MON87427: Performance assessment based on the metrologically traceable reference values as assigned values

The Asia Pacific Metrology Program and the Accreditation Cooperation joint Proficiency Testing (PT) program for the quantification of genetically modified maize MON87427 was organized by the National Institute of Metrology, China, to enhance the measurement accuracy and metrological traceability in the region. Certified reference materials were employed as test samples; metrologically traceable certified reference values served as PT reference values (PTRVs) for evaluating the participants results. The consensus values obtained from the participants were higher than the assigned values, potentially due to the systematic effects of DNA extraction process. The participants' relatively poor overall performance by the ζ-score compared with z-score demonstrates their need to thoroughly investigate quantification bias to elevate the measurement capability of genetically modified (GM) content and deepen their understanding of uncertainty estimation. This program confirmed the importance of using metrologically traceable reference values instead of consensus values as PTRV for reliable performance assessment.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument

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.

Isolation, characterization and identification of Klebsiella sp. dor_27f isolated from textile effluents

This research study aimed to isolate and characterize a new bacterial strain from textile effluents. To do this, bacteria were cultured using the MSM medium, where a colony was isolated through six successive pickings. It then underwent a DNA extraction process using the phenol-chloroform-isoamyl alcohol methodology, and an electrophoresis was carried out to confirm the extraction. In addition, the isolated colony was identified as Klebsiella sp. by sequencing the 16S rRNA gene using bioinformatics tools. To observe its biotechnological potential, the bacterial strain was grown in an MSM broth enriched with Golden Yellow K2R azoic dye at a concentration of 50 mg/L, showing a percentage of decolorization of 74 % after 72 hours at 37 °C, indicating the potential of the isolated colony for the development of bio-remediation processes for effluents containing azoic dyes. Keywords: Bacterial strain, Klebsiella sp., DNA extraction, decolorization, effluents.

Virtual screening and site-directed mutagenesis-derived aptamers for precise Salmonella typhimurium prediction: emphasizing OmpD targeting and G-quadruplex stability

The efficient detection of the foodborne pathogen Salmonella typhimurium has historically been hampered by the constraints of traditional methods, characterized by protracted culture periods and intricate DNA extraction processes for PCR. To address this, our research innovatively focuses on the crucial and relatively uncharted virulence factor, the Outer Membrane Protein D (OmpD) in Salmonella typhimurium. By harmoniously integrating the power of virtual screening and site-directed mutagenesis, we unveiled aptamers exhibiting marked specificity for OmpD. Among these, aptamer 7ZQS stands out with its heightened binding affinity. Capitalizing on this foundation, we further engineered a repertoire of mutant aptamers, wherein APT6 distinguished itself, reflecting unmatched stability and specificity. Our rigorous validation, underpinned by cutting-edge bioinformatics tools, amplifies the prowess of APT6 in discerning and binding OmpD across an array of Salmonella typhimurium strains. This study illuminates a transformative approach to the prompt and accurate detection of Salmonella typhimurium, potentially redefining boundaries in applied analytical chemistry and bolstering diagnostic precision across diverse research and clinical domains. Communicated by Ramaswamy H. Sarma

Thread-powered cell lysis and isotachophoresis: unlocking microbial DNA for diverse molecular applications.

Central to the domain of molecular biology resides the foundational process of DNA extraction and purification, a cornerstone underpinning a myriad of pivotal applications. In this research, we introduce a DNA extraction and purification technique leveraging polypropylene (PP) threads. The process commences with robust cell lysis achieved through the vigorous agitation of interwoven PP threads. The friction between the threads facilitates cell lysis especially those microbes having tough cell wall. For purification of DNA, thread-based isotachophoresis was employed which makes the whole process swift and cost-effective. Lysed cell-laden threads were submerged in a trailing electrolyte which separated DNA from other cellular contents. The process was performed with a tailored ITP device. An electric field directs DNA, cell debris, trailing electrolyte, and leading electrolyte toward the anode. Distinct ion migration resulted in DNA concentrating on the PP thread's anode-proximal region. The SYBR green dye is used to visualize DNA as a prominent green zone under blue light. The purified DNA exhibits high purity levels of 1.82 ± 0.1 (A260/A280), making it suitable for various applications aiming at nucleic acid detection.

Preserving morphology while extracting DNA: a non-destructive field-to-museum protocol for slide-mounted specimens.

Our study aimed to develop an optimised laboratory protocol ensuring the preservation of morphological structures and extraction of high-quality DNA sequences from Psychodidae (Insecta, Diptera) specimens. With 310 analysed specimens, we investigated the impact of distinct laboratory treatments by employing two shaking categories (constant and interrupted) with five different incubation periods (16, 12, 8, 4 and 2 hours) during the DNA extraction process. Notably, 80.65% of the specimens exhibited morphological changes during DNA extraction. Our results indicated no statistical difference between constant and interrupted shaking for the total of morphological structures lost. However, within each shaking category, the loss of structures was influenced significantly by the incubation period. Prolonged incubation correlated with increased structural losses, whereas shorter incubation periods caused minor alterations in structures lost. In addition, our results showed a significant difference between constant and interrupted shaking treatments for DNA concentration. Likewise, the incubation period showed differences within each shaking category. Successful COI sequencing was achieved in 89.6% of specimens, with negligible differences in DNA fragment lengths across treatments. Our findings underscore the importance of an optimised protocol and its potential in systematic research involving nematoceran dipteran specimens by balancing morphological integrity and DNA extraction efficiency.

3-hour genome sequencing and targeted analysis to rapidly assess genetic risk

PurposeRapid genetic testing in the critical care setting may guide diagnostic evaluation, direct therapies, and help families and care providers make informed decisions about goals of care. We tested whether a simplified DNA extraction and library preparation process would enable us to perform ultrarapid assessment of genetic risk for a Mendelian condition, based on information from an affected sibling, using long-read genome sequencing and targeted analysis. MethodsFollowing extraction of DNA from cord blood and rapid library preparation, genome sequencing was performed on an Oxford Nanopore PromethION. FASTQ files were generated from original sequencing data in near real-time and aligned to a reference genome. Variant calling and analysis were performed at timed intervals. ResultsWe optimized the DNA extraction and library preparation methods to create sufficient library for sequencing from 500 μL of blood. Real-time, targeted analysis was performed to determine that the newborn was neither affected nor a heterozygote for variants underlying a Mendelian condition. Phasing of the target region and prior knowledge of the affected haplotypes supported our interpretation despite a low level of coverage at 3 hours of life. ConclusionThis proof-of-concept experiment demonstrates how prior knowledge of haplotype structure or familial variants can be used to rapidly evaluate an individual at risk for a genetic disease. Although ultrarapid sequencing remains both complex and cost prohibitive, our method is more easily automated than prior approaches and uses smaller volumes of blood and thus may be more easily adopted for future studies of ultrarapid genome sequencing in the clinical setting.

Challenges and Innovations in Forensic Identification: Case Study of Decomposed Remains in a Crocodile's Body for Missing Child Identification

Identifying a missing child from decomposed remains in a crocodile's body poses significant challenges and complexity. Forensic investigations involving human remains within animal’s body can present unique difficulties apart from conventional investigative scenarios. Decomposed remains added an additional layer of difficulty as the digestive process of a crocodile can significantly degrade the body and its features. Forensic anthropology examinations were impeded due to the limited availability of tiny bone samples. In this scenario decomposed bone pieces were the only remaining material for DNA analysis, the choice of an efficient DNA recovery procedure becomes crucial for successful outcome. In the study, prior to the DNA extraction process, a decontamination technique was employed, followed by decalcification method on the bone sample. The resulting, clean and decalcified bone sample was then subjected to extraction using a silica-based method, and the extracted DNA was subsequently typed through STR amplification. The derived DNA profile information from the bone sample pertaining to the child is compared with the profile details of parents for identification purposes.

Induction of Artesunate Resistance in Plasmodium falciparum 3D7 Strain Using Intermittent Exposure Method and Comparing P.fk13 Sequence between Susceptible and Resistant Strains.

Resistance to artemisinin has threatened major achievements in malaria control, more investigations is needed about resistant strains and related genes. We aimed to induce resistance to artesunate in the Plasmodium falciparum 3D7 strain using intermittent exposure method and comparing P.fk13 gene sequence between susceptible and resistance strains. P. falciparum 3D7 strain was cultured according to Trager & Jensen method with some modifications. Serial concentrations between 10-2 mol/l, to 10-7mol/l were prepared, then P. falciparum 3D7 was exposed to each of the dilution to determine IC50 and lethal dose. Sensitivity reduction process was started from the concentration of 10-7mol/l and ended at 10-2mol/l. Exposed parasites were collected after at least 27 days after cultivation in each drug concentration. DNA extraction, PCR and sequencing process were performed to investigate any possible mutations in the P.fk13 gene sequence. Effectiveness of 10-2mol/l concentration of artemisinin was found as a lethal dose. IC50 value was equal to 5×10-4 mol/l. The resistant strain was provided in the lab, sequenced and registered in the gene bank as P.f Art -2, (accession number MH796123. 1). Alignment of this registered sample showed no mutation in P.f kelch13 gene in comparison with standard strain submitted in the GenBank. Resistance to artesunate in malaria parasite may occur but with no mutation in the P.f kelch13 gene. Therefore, whole genome sequencing should be applied to determine mutations in resistant strains.

New polymer brush-coated monodisperse magnetic nanoparticles prepared via interface-mediated RAFT polymerization for high-throughput DNA extraction from pathogen bacteria

Polymerase chain reaction (PCR) is gold-standard method for molecular detection of pathogens. DNA extraction from a complex biological medium is a crucial process for PCR based detection. Recently, magnetic nanoparticles-based methods are of great interest for genetic material isolation. In our work, we offer new magnetic nanoparticles based high-throughput DNA extraction method for pathogen detection in biofluids. For this purpose, first time polymer brush coated monodisperse magnetic nanoparticles were prepared with interface-mediated RAFT polymerization to be used in DNA extraction process. After synthesizing Fe3O4 nanoparticles, silica shell was coated on the magnetic nanoparticle core. Polymer brushes was immobilized on the amine functionalized silica layer using RAFT agent. Subsequently, the resulting particles with poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) grafted were finalized through interface-mediated RAFT polymerization. The functional groups on the nanoparticles were characterized using FTIR spectroscopy. Size distribution and zeta potential analysis of the particles were performed by TEM and Zeta-sizer respectively. Adsorption and recovery studies of plasmid DNA were carried out on the polymer-coated magnetic nanoparticles. The developed particle provided both the rapid extraction of DNA and high-yield DNA recovery without requiring extended incubation and elution time. The maximum adsorption capacity was determined as 238.1 μg/mg. About 90 % of the input DNA was recovered. The total time of DNA extraction took just 20 min. We have successfully demonstrated the extraction of dsDNA of Enterococcus faecalis spiked to reference blood sample using the developed magnetic particles. The extracted plasmid DNA and dsDNA were confirmed by gel electrophoresis and qPCR methods respectively. The results showed that the proposed magnetic iron oxide nanoparticles (MIONp) will be a strong candidate for DNA extraction process from biological matrices.

Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology.We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction.DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1µg/mL, 100ng/mL, 10ng/mL and 5ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.