Assessment of Blood and Semen Detection and DNA Collection from Swabs up to Three Months after Deposition on Five Different Cloth Materials.

Body fluid identification plays a crucial role in criminal investigations. Because of their presence in many cases, blood and semen are the most relevant body fluids in forensic sciences. Based on antigen-antibody reactions binding unique proteins for each body fluid, serological assays represent one of the most rapid and highly specific tests for blood and semen. Currently, few studies have assessed the factors affecting body fluid identification by applying these assays. This work aimed to study the effect of different fabrics from clothes and time since deposition on identification through immunochromatographic tests for blood and semen, DNA isolation, and STR profiling from these samples. Body fluids were deposited on black- and white-dyed denim and cotton fabrics, and on leather. Afterward, blood and semen were sampled at 1 day, 30 days, and 90 days after deposition and identified by using the SERATEC® HemDirect Hemoglobin Test and the PSA Semiquant and SERATEC® BLOOD CS and SEMEN CS tests, respectively. Laboratory and crime scene tests presented similar performances for the detection of blood and semen stains on every tested fabric. No differences were found on band intensities between timepoints for all fabrics. It was possible to recover and identify blood and semen samples up to three months after deposition and to obtain full STR profiles from all the tested fabrics. Both body fluid STR profiles showed differences in their quality between 1 and 90 days after deposition for all fabrics except for black cotton for semen samples. Future research will expand the results, assessing body fluid identification on other substrates and under different environmental conditions.

Quality assessment of enzymatic methyl-seq library constructed using crude cell lysate.

Enzymatic methyl-seq (EM-seq), an enzyme-based method, identifies genome-wide DNA methylation, which enables us to obtain reliable methylome data from purified genomic DNA by avoiding bisulfite-induced DNA damage. However, the loss of DNA during purification hinders the methylome analysis of limited samples. The crude DNA extraction method is the quickest and minimal sample loss approach for obtaining useable DNA without requiring additional dissolution and purification. However, it remains unclear whether crude DNA can be used directly for EM-seq library construction. In this study, we aimed to assess the quality of EM-seq libraries prepared directly using crude DNA. The crude DNA-derived libraries provided appropriate fragment sizes and concentrations for sequencing similar to those of the purified DNA-derived libraries. However, the sequencing results of crude samples exhibited lower reference sequence mapping efficiencies than those of the purified samples. Additionally, the lower-input crude DNA-derived sample exhibited a marginally lower cytosine-to-thymine conversion efficiency and hypermethylated pattern around gene regulatory elements than the higher-input crude DNA- or purified DNA-derived samples. In contrast, the methylation profiles of the crude and purified samples exhibited a significant correlation. Our findings indicate that crude DNA can be used as a raw material for EM-seq library construction.

Comprehensive assessment of 12 commercial DNA-binding dyes as alternatives to ethidium bromide for agarose gel electrophoresis.

Staining and visualization of the nucleic acid bands on agarose gels using ethidium bromide (EB) has been a widely used technique in molecular biology. Although it is an efficient dye for this purpose, EB is known to be mutagenic and genotoxic in humans. This led to the emergence of various alternative dyes, which were claimed to be safer and more efficient than EB. However, these dyes portray varied sensitivity and interference with the electrophoretic mobility of nucleic acids. This work aimed at assessing ten nucleic acid-binding dyes and two prestained dyes for these properties by three staining techniques, such as precasting, preloading, and poststaining. Of these, preloading was not suitable for any of the dye while poststaining worked optimal for most of them. Precasting was suitable for only four dyes viz. DNA Stain G, SYBR™ safe, EZ-Vision® in-gel, and LabSafe™. Poststaining was, in general, a costlier method than precasting. The work gives a comprehensive understanding of the performance of nucleic acid-binding dyes for routine molecular biology experiments.

Assessment of measurement accuracy of amplified DNA using a colorimetric loop-mediated isothermal amplification assay.

A colorimetric loop-mediated isothermal amplification assay detects changes in pH during amplification based on color changes at a constant temperature. Currently, various studies have focused on developing and assessing molecular point-of-care testing instruments. In this study, we evaluated amplified DNA concentrations measured using the colorimetric LAMP assay of the 1POT™ Professional device (1drop Inc, Korea). Results of the 1POT analysis of clinical samples were compared with measurements obtained from the Qubit™ 4 and NanoDrop™ 2000 devices (both from Thermo Fisher Scientific, MA, USA). These results showed a correlation of 0.98 (95% CI: 0.96-0.99) and 0.96 (95% CI: 0.92-0.98) between 1POT and the Qubit and NanoDrop. 1POT can measure amplified DNA accurately and is suitable for on-site molecular diagnostics.

High-Resolution Multiparameter DNA Flow Cytometry for Accurate Ploidy Assessment and the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin-Embedded Tissues.

This article contains detailed protocols for the simultaneous flow cytometric identification of tumor cells and stromal cells and measurement of DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for accurate DNA content assessments of FFPE carcinoma tissues. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 (near-haploidy) and of keratin-positive tumor cells with a DNA index close to 1.0 in overall DNA aneuploid samples, thus improving DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiparameter DNA content analysis of FFPE carcinomas Alternate Protocol 1: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue and red excitation Alternate Protocol 2: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue excitation Support Protocol: Sorting cell population from FFPE carcinomas.

Precise, wide field, and low-cost imaging and analysis of core-shell beads for digital polymerase chain reaction.

Digital droplet reactors have become a valuable tool for the analysis of single cells, organisms, or molecules by discretising reagents into picolitre or nanolitre volumes. However, DNA-based assays typically require processing of samples on the scale of tens of microlitres, with the detection of as few as one or as many as a hundred thousand fragments. Through the present work, we introduce a flow-focusing microfluidic device that produces 120 picolitre core-shell beads, which are assembled into a monolayer in a Petri dish for visualization and analysis. The bead assembly is subjected to polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the DNA concentration of the sample. We use a low-cost 21-megapixel digital camera and macro lens to capture wide-field fluorescence images with a 10-30 mm2 field-of-view at magnifications ranging from 5× to 2.5×. A customised Python script analysed the acquired images. Our study demonstrates the ability to perform digital PCR analysis of the entire bead assembly through end-point imaging and compare the results with those obtained through RT-qPCR.

A cost-benefit analysis for use of large SNP panels and high throughput typing for forensic investigative genetic genealogy.

Next-generation sequencing (NGS), also known as massively sequencing, enables large dense SNP panel analyses which generate the genetic component of forensic investigative genetic genealogy (FIGG). While the costs of implementing large SNP panel analyses into the laboratory system may seem high and daunting, the benefits of the technology may more than justify the investment. To determine if an infrastructural investment in public laboratories and using large SNP panel analyses would reap substantial benefits to society, a cost-benefit analysis (CBA) was performed. This CBA applied the logic that an increase of DNA profile uploads to a DNA database due to a sheer increase in number of markers and a greater sensitivity of detection afforded with NGS and a higher hit/association rate due to large SNP/kinship resolution and genealogy will increase investigative leads, will be more effective for identifying recidivists which in turn reduces future victims of crime, and will bring greater safety and security to communities. Analyses were performed for worst case/best case scenarios as well as by simulation sampling the range spaces with multiple input values simultaneously to generate best estimate summary statistics. This study shows that the benefits, both tangible and intangible, over the lifetime of an advanced database system would be huge and can be projected to be for less than $1 billion per year (over a 10-year period) investment can reap on average > $4.8 billion in tangible and intangible cost-benefits per year. More importantly, on average > 50,000 individuals need not become victims if FIGG were employed, assuming investigative associations generated were acted upon. The benefit to society is immense making the laboratory investment a nominal cost. The benefits likely are underestimated herein. There is latitude in the estimated costs, and even if they were doubled or tripled, there would still be substantial benefits gained with a FIGG-based approach. While the data used in this CBA are US centric (primarily because data were readily accessible), the model is generalizable and could be used by other jurisdictions to perform relevant and representative CBAs.

Shedder status-An analysis over time and assessment of various contributing factors.

The shedder status of a person is an important consideration when evaluating probabilities of DNA transfer during activity-level assessments. As an extension of our previously published study, the shedder statuses of 38 individuals were reassessed 1 year later. The study found that shedder status may change over time for some individuals and was associated with one's gender, number of items touched, and mobile phone usage. In 29% of touch events, no DNA allele was detected and in 99% of touch events, the amount of DNA deposited was <2 ng. The study also found that in 0.6% of touch events, the participant could be excluded as a contributor of the observed DNA profile, with another person being included. Additionally, our investigations suggest that the current three-category system for shedder status classification may require further refinement to better represent the individuals' shedder status in a population.

Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers.

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.

An assessment of DNA extraction methods from blood-stained soil in forensic science.

In forensic crime scene investigations, biological fluids such as blood are commonly found in soil. However, the analysis of blood-stained soil can be challenging due to the presence of inhibitors which limit the effective extraction and amplification of the deoxyribonucleic acid (DNA) required to produce a reportable DNA profile. There are some extraction methods that have been applied to blood-stained soil in forensic science, but these have produced sporadic results. This research has taken a number of different extraction methods from the fields of ancient DNA and environmental DNA and broken them down into the individual steps of pre-treatment, incubation, separation and purification. These steps were assessed independently then combined into various extraction methods to determine the best technique that can effectively and reliably profile human DNA from blood-stained soil. Testing involved assessment of three extraction buffers, (cetyltrimethylammonium bromide, guanidine thiocyanate, and proteinase K), four pre-treatment methods, (polyvinylpyrrolidone, ethylenediaminetetraacetic acid, hydrochloric acid, and sodium hydroxide), three separation steps, (centrifugation, phenol chloroform, and chloroform) and four purification steps, (size exclusion chromatography, bind elute columns, isopropanol precipitation and silica magnetic beads). The most effective procedure was found to be a polyvinylpyrrolidone pre-treatment with a proteinase K extraction buffer followed by magnetic silica bead purification with or without centrifugation. However, centrifugation separation was found to be equally effective after the pre-treatment step as after the incubation step. Our results shows that most of the current forensic procedures would benefit from the addition of a pre-treatment step prior to processing through the automated DNA profiling pipeline.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

OBJECTIVES: Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. METHODS: Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. CONCLUSIONS: The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.

Assessment of Multiple Annealing and Looping-Based Amplification Cycle-Based Whole-Genome Amplification for Short Tandem Repeat Genotyping of Low Copy Number-DNA.

Aim: A common problem in forensic practice is the lack of sufficient amounts of good quality genomic DNA. A possible solution is the amplification of the available genomic DNA before locus-specific polymerase chain reaction (PCR) analysis. The aim of this study was to evaluate multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) for short tandem repeat (STR) genotyping of low copy number DNA (LCN-DNA). Materials and Methods: DNA isolated from five blood samples was quantified and diluted to 250, 150, 100, 50, 25, and 5 pg/µL. After preamplification with MALBAC, WGA products were quantified. PCR-STR genotyping was performed in triplicate using dilution or purification-treated WGA products for each level of DNA. STR profiles were analyzed and compared with that from non-WGA DNA. Results: The purification treatment performed better than dilution of the MALBAC-based WGA products. Compared with the non-WGA DNA, both the average number and peak heights of correct alleles were significantly improved after preamplification with the MALBAC-based WGA at DNA inputs of ≤50 pg. Like other WGA methods, allele dropout and allele drop-in were observed in the profiling results for many samples. Conclusions: MALBAC shows great potential in LCN-DNA analysis and could find broader application in the fields of forensics and genetics.

Low-cost Imaging of Fluorescent DNA in Agarose Gel Electrophoresis using Raspberry Pi cameras.

Low-cost analytical solutions built around microcomputers like the Raspberry Pi help to facilitate laboratory investigations in resource limited venues. Here, three camera modules (V1.3 with and without filter, as well as NoIR) that work with this microcomputer were assessed for their suitability in imaging fluorescent DNA following agarose gel electrophoresis. Evaluation of their utility was based on signal-to-noise (SNR) and noise variance metrics that were developed. Experiments conducted with samples were subjected to Polymerase Chain Reaction (PCR), and the amplified products were separated using gel electrophoresis and stained with Midori green. Image analysis revealed the NoIR camera performed the best with SNR and noise variance values of 21.7 and 0.222 respectively. In experiments conducted using UV LED lighting to simulate ethidium bromide (EtBr) excitation, the NoIR and V1.3 with filter removed cameras showed comparable SNR values.

Assessment of cytotoxicity, genotoxicity, and mutagenicity of the dexchlorpheniramine reference standard and pharmaceutical formula in human peripheral blood mononuclear cells

Abstract Dexchlorpheniramine is a first-generation classical antihistamine, clinically used to treat allergies. The main objective of our study was to evaluate the effects of the dexchlorpheniramine reference standard (DCPA Ref. St) and a pharmaceutical formula on DNA in human peripheral blood mononuclear cells (PBMCs). We exposed PBMCs to five different concentrations (0.5, 2.5, 5, 10, and 50 ng/mL) of DCPA Ref. St DCPA Ref. St and pharmaceutical formula in order to evaluate their cytotoxic, genotoxic, and mutagenic potential. The results showed that both dexchlorpheniramine formulations did not affect PBMC viability and CD3+, CD4+, or CD8+ lymphocyte subpopulations. The DCPA Ref. St and pharmaceutical formula neither induced genotoxic or mutagenic effects nor numerical or structural chromosomal alterations in PBMCs after 24 hours of exposure.

Comparative assessment of antimicrobial, antiradical and cytotoxic activities of cannabidiol and its propyl analogue cannabidivarin.

Cannabidiol and cannabidivarin are phytocannabinoids produced by Cannabis indica and Cannabis sativa. Cannabidiol has been studied more extensively than its propyl analogue cannabidivarin. Therefore, we performed a battery of in vitro biological assays to compare the cytotoxic, antiradical and antibacterial activities of both cannabinoids. Potential mitochondrial metabolism alterations, DNA synthesis inhibition, and plasma membrane damage were studied by MTT assay, BrdU-ELISA and LDH assay of cancer and normal human cells exposed to cannabinoids. ABTS and DPPH assays were performed to observe the effects of the cannabinoids on free radicals. Microbial susceptibility tests were performed to study the activity of the cannabinoids in two bacterial species implicated in human infections, Escherichia coli and Staphylococcus aureus. The results showed that the cannabinoids induced medium levels of cytotoxicity in cancer and normal cells at concentrations ranging from 15.80 to 48.63 and from 31.89 to 151.70 µM, respectively, after 72 h of exposure. Cannabinoids did not exhibit a strong antioxidant capacity in scavenging ABTS or DPPH radicals. No evident differences were observed between the two cannabinoids in antimicrobial activity, except with respect to S. aureus, which showed greater susceptibility to cannabidiol than to cannabidivarin after 72 h of exposure.

Assessment of Metastatic Colorectal Cancer (CRC) Tissues for Interpreting Genetic Data in Forensic Science by Applying 16 STR Loci among Saudi Patients.

BACKGROUND: In forensic science, there are cases when the only available provider of biological data is samples of malignant tissues. It can be useful in identification and/or paternity tests. Still, such samples have ambiguities because of microsatellite instability (MSI) and loss of heterozygosity (LOH) effects, being often related to neoplasia. METHODS: This research evaluates 16 autosomal short tandem repeat (STR) loci (traditional in forensic investigations) to get genetic data. MSI and LOH were estimated in DNA patterns derived from 73 Saudi respondents (30 healthy individuals and 43 persons with diagnosed colorectal cancer (CRC). Upon deriving DNA from blood, CRC specimens were obtained in both groups, along with the adjoining normal non-cancerous tissues (N-CRC). All specimens and 16 loci (15 STR loci and Amelogenin) were evaluated. Moreover, both colorectal samples were histologically analyzed utilizing HandE staining. RESULTS: Findings revealed non-essential variability in genetic information because of MSI and/or LOH. In CRC, mutations rates were 0.42% (MSI) and 1.62% (LOH). In N-CRC, mutation rates were 0.00% (MSI) and 0.59% (LOH). Further, LOH-related deviations were recorded in 5 loci out of 16. MSI-related deviations were recorded in 4 out of 16 loci, being present in CRC samples only. Genetic deviations within the marker loci might inform about false homozygosity/heterozygosity. Similarly, false gender might come from improper interpretation of DNA profiles. Finally, histopathological trials showed considerable histopathological alterations contrasted to N-CRC. CONCLUSION: This study is unique in demonstrating the application of 16 autosomal STRs from CRC samples and their comparison with the adjoining N-CRCs in Saudi participants, contributing to the field of forensic science. The experiment revealed no considerable distinctions, while showing that cancer tissues might display MSI and LOH effects that might challenge data interpretation, if STRs are to be applied in the forensic investigation.

The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition.

We present a method for the evaluation of fluorescence fluctuations on the basis of Mandel's Q parameter, using sampling time-dependent factorial cumulants. By relating the Q parameter to the sampling time, we obtain the mean single molecule rate (mSMR), an easy to interpret expression that provides both brightness and diffusion information. The model is suitable for the widely used confocal setups with single photon excitation and a single detection channel. We present a way to correct the mSMR for afterpulsing, dead time and background noise. To account for photokinetic effects at short sampling times, we expand the model by a simple on/off isomerization term, which is similar to the well-known triplet model. The functionality of the mSMR is shown using Monte Carlo simulations. The correction mechanisms and the experimental applicability of the model are then demonstrated by DNA measurements of defined composition. By systematically analyzing DNA mixtures, we can show that at large sampling times, the mSMR correctly describes the single molecule brightness rates and the diffusive properties of DNA molecules. At short sampling times, the photokinetic effects of isomerization are accurately described by the mSMR model. Since additionally the mSMR can easily be corrected for measurement artefacts such as detector dead time, afterpulsing and background noise, this is a valuable advantage over the standard method of fluorescence correlation spectroscopy.

Utilization of a Colorectal Cancer Screening Test Among Individuals With Average Risk.

Importance: Colorectal cancer (CRC) screening reduces CRC incidence and mortality. It is important to examine screening patterns over time, including after the introduction of new screening modalities. Objective: To compare use of CRC screening tests before and after the availability of the multitarget stool DNA (mt-sDNA) test, given that endorsed options have changed. Design, Setting, and Participants: This longitudinal cohort study used administrative claims data to examine CRC screening use in 2 discrete periods: before (August 1, 2011, to July 31, 2014) and after (August 1, 2016, to July 31, 2019) the mt-sDNA test became available. The MarketScan Commercial and Medicare Supplemental databases were queried for individuals aged 45 to 75 years between August 1, 2011, and July 31, 2019, with average risk of CRC and with continuous enrollment in the databases from August 1, 2001, to July 31, 2019. Main Outcomes and Measures: The proportion of individuals up to date or not due for CRC screening during each measurement year and the type of screening test used among individuals due for screening. Data were reported overall and among individuals aged 45 to 49 or 50 years and older on August 1, 2011. Results: A total of 97 776 individuals with average risk were identified. Individuals had a mean (SD) age of 50.8 (3.5) years, and 54 227 (55.5%) were women. The proportion of individuals with average risk aged 50 to 75 years with commercial or Medicare supplemental insurance who were up to date with CRC screening increased from 50.4% in 2011 (30 605 of 60 770) to 69.7% in 2019 (42 367 of 60 770). Among individuals due for screening and screened, the use of high-sensitivity fecal occult blood test (FOBT) decreased between 2011 (1088 of 6241 eligible individuals [17.7%]) and 2019 (195 of 2943 eligible individuals [6.6%]), and the use of mt-sDNA increased between 2016 (58 of 3014 eligible individuals [1.9%]) and 2019 (418 of 2943 eligible individuals [14.2%]). No consistent trends were observed with fecal immunochemical test (FIT) or screening colonoscopy. Computed tomography colonography, double-contrast barium enema, and flexible sigmoidoscopy were rarely performed. Conclusions and Relevance: In this cohort study, the proportion of individuals with average risk who were up to date with CRC screening increased between 2011 and 2019 but remained suboptimal. There were no substantial changes in the use of the colonoscopy or FIT; however, there was an increase in the adoption of mt-sDNA and a decrease in the use of FOBT during the study period.

Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal.

The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2'-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.