DNA Stability Research Articles

Serum Zinc Concentration in Patients with Alopecia Areata.

Alopecia areata is an autoimmune non-scarring disease in which the exact mechanism that induces loss of immune privilege is unknown. Zinc is important for DNA stability and repair mechanisms that are essential in maintaining normal hair growth. Zinc deficiency has been investigated as an important factor in many autoimmune diseases, and may have a possible role in the aetiopathogenesis of alopecia areata. This study included 32 patients with severe forms of alopecia areata, and 32 age- and sex-matched healthy controls. When comparing serum zinc levels in these 2 groups, statistically significantly lower zinc concentrations were found in the alopecia areata group (p = 0.017). Detected zinc deficiency was statistically more prevalent in patients with alopecia areata (p = 0.011). Evaluating patients with alopecia areata, a statistically significant negative correlation between serum zinc levels and severity of the disease was found (ρ = 0.006). The results indicate that zinc serum assessment is necessary in patients with alopecia areata. Low serum zinc levels were found to correlate with severity of alopecia areata. Given that most severe forms of alopecia areata are frequently most treatment-resistant, additional randomized control trials examining zinc supplementation are necessary to investigate its potential role in the restoration of hair follicles.

DNA integrity under alkaline conditions: An investigation of factors affecting the comet assay.

The effect of pH on DNA integrity was assessed using a three-step approach. The comet assay was used on a whole genome level, with three different protocols: neutral (no alkaline unwinding), flash (pH 12.5 with 2.5min unwinding), and the conventional alkaline protocol (pH>13 with 40min unwinding). Real-time quantitative PCR (RT-qPCR) was then used to study the isolated DNA, revealing that gene amplification decreased with increasing pH, indicating DNA degradation. Specially designed molecular beacons were used to examine DNA at the molecular level, with or without alkali-labile site (ALS) insertions. At pH 12.5, fluorescence in the hairpins with ALS started to increase after 30min, while at pH>13, this increase was already observed after 5min, indicating a significant increase in DNA strand breaks. Liquid chromatography analysis was also used, demonstrating that the hairpins remained intact up to pH 10, even after 1h exposure, whereas, at pH 12.5, partial conversion into strand breaks occurred after 30min. At pH>13, the hairpins were almost completely degraded after 30min. The flash protocol effectively detects DNA single- and double-strand breaks and identified these damages after 2.5min of alkaline treatment at pH 12.5. When the hairpins were exposed to pH 12.5 for 60min, ALS were converted to strand breaks, demonstrating the sensitivity of this approach to detect changes in DNA structure. These findings indicate that pH poses a substantial risk to DNA integrity, leading to significantly higher background levels of DNA damage compared to conditions closer to neutrality. Our study demonstrates the importance of understanding the influence of pH on DNA stability and provides insights into risks associated with alkaline environments, especially at pH>13.

Scurvy: A treatable forgotten fatal differential diagnosis and potential etiology of leukemia and aplastic anemia in pediatric population.

Scurvy is a rare nutritional deficiency disease which is less likely to be suspected and it mostly lead to delayed diagnosis. It can present with features which can mislead clinicians to misdiagnose the condition as leukemia or aplastic anemia. This can subject patients to the wrong management which leads to poor outcome and increased preventable morbidity and mortality. Vitamin C deficiency is still prevalent among pediatric population even in the modern days and should no longer be considered as historical condition. Chromosomal fragility has been greatly accounted for the development of leukemia and aplastic anemia secondary to various triggers. The role of vitamin C toward DNA stability, prevention, and control of mutations have been documented. Vitamin C plays a vital role in hematopoiesis by controlling regulation and prevent dysfunction of hematopoietic stem cells. Scurvy deficiency has been a silent growing clinical problem which needs a high index of suspicion for a clinician to pick it. It should be considered as one among potential differential diagnosis of leukemia and aplastic anemia especially in the pediatric population. History of any dietary restriction should be obtained and addressed properly. Serum vitamin C should be among the essential laboratory workout in diagnosis of both leukemia and aplastic anemia. All patients suspected to have such conditions should be screened and supplemented for vitamin C deficiency irrespective of positive confirmatory test results of leukemia or aplastic anemia since the probability of co-occurrence is likely also. Moreover, studies should be conducted to explore the clinical link, if any, between vitamin C deficiency or insufficiency and development of leukemia and aplastic anemia among the pediatric population given its physiological and genomic role in hematopoiesis. Furthermore, the potential pharmacological therapeutic use of vitamin C in treatment of leukemia and aplastic anemia should be determined clinically.

Identification of DNA N4-methylcytosine Sites via Multiview Kernel Sparse Representation Model

Identifying DNA N4-methylcytosine (4mC) sites is of great significance in biological research, such as chromatin structure, DNA stability, DNA-protein interaction and controlling gene expression. However, the traditional sequencing technology to identify 4mC sites is very time-consuming. In order to detect 4mC sites, we develop a multi-view learning method for achieving more effectively via merging multiple feature spaces. Furthermore, we think about whether the multi-view learning method can improve the across species classification ability by fusing data of multiple species. In our study, we propose a multi-view Laplacian kernel sparse representation-based classifier, called MvLapKSRC-HSIC. First, we make use of three feature extraction methods (PSTNP, NCP, DPP) to extract the DNA sequence features. MvLapKSRC-HSIC uses a kernel sparse representation-based classifier with graph regularization. In order to maintain the independence between various views, we add a multi-view regularization term constructed by Hilbert-Schmidt independence criterion (HSIC). In the experiments, MvLapKSRC-HSIC is applied on six datasets, so as to compare with other popular methods in single species and cross-species experiments. All experimental results show that MvLapKSRC-HSIC is superior to other outstanding methods on both single species and cross-species. Importantly, MvLapKSRC-HSIC can identify a series of potential DNA 4mC sites, which have not yet been experimentally evaluate on multiple species and merit further research.

Single-Nucleotide Polymorphisms in Genes Maintaining the Stability of Mitochondrial DNA Affect the Occurrence, Onset, Severity and Treatment of Major Depressive Disorder.

One of the key features of major depressive disorder (MDD, depression) is increased oxidative stress manifested by elevated levels of mtROS, a hallmark of mitochondrial dysfunction, which can arise from mitochondrial DNA (mtDNA) damage. Thus, the current study explores possibility that the single-nucleotide polymorphisms (SNPs) of genes encoding the three enzymes that are thought to be implicated in the replication, repair or degradation of mtDNA, i.e., POLG, ENDOG and EXOG, have an impact on the occurrence, onset, severity and treatment of MDD. Five SNPs were selected: EXOG c.-188T > G (rs9838614), EXOG c.*627G > A (rs1065800), POLG c.-1370T > A (rs1054875), ENDOG c.-394T > C (rs2977998) and ENDOG c.-220C > T (rs2997922), while genotyping was performed on 538 DNA samples (277 cases and 261 controls) using TaqMan probes. All SNPs of EXOG and ENDOG modulated the risk of depression, but the strongest effect was observed for rs1065800, while rs9838614 and rs2977998 indicate that they might influence the severity of symptoms, and, to a lesser extent, treatment effectiveness. Although the SNP located in POLG did not affect occurrence of the disease, the result suggests that it may influence the onset and treatment outcome. These findings further support the hypothesis that mtDNA damage and impairment in its metabolism play a crucial role not only in the development, but also in the treatment of depression.

Highly Uniform DNA Monolayers Generated by Freezing‐Directed Assembly on Gold Surfaces Enable Robust Electrochemical Sensing in Whole Blood

AbstractAssembling DNA on solid surfaces is fundamental to surface‐based DNA technology. However, precise control over DNA conformation and organization at solid–liquid interfaces remains a challenge, resulting in limited stability and sensitivity in biosensing applications. We herein communicate a simple and robust method for creating highly uniform DNA monolayers on gold surfaces by a freeze‐thawing process. Using Raman spectroscopy, fluorescent imaging, and square wave voltammetry, we demonstrate that thiolated DNA is concentrated and immobilized on gold surfaces with an upright conformation. Moreover, our results reveal that the freezing‐induced DNA surfaces are more uniform, leading to improved DNA stability and target recognition. Lastly, we demonstrate the successful detection of a model drug in undiluted whole blood while mitigating the effects of biofouling. Our work not only provides a simple approach to tailor the DNA‐gold surface for biosensors but also sheds light on the unique behavior of DNA oligonucleotides upon freezing on the liquid‐solid interface.

Highly Uniform DNA Monolayers Generated by Freezing-Directed Assembly on Gold Surfaces Enable Robust Electrochemical Sensing in Whole Blood.

Assembling DNA on solid surfaces is fundamental to surface-based DNA technology. However, precise control over DNA conformation and organization at solid-liquid interfaces remains a challenge, resulting in limited stability and sensitivity in biosensing applications. We herein communicate a simple and robust method for creating highly uniform DNA monolayers on gold surfaces by a freeze-thawing process. Using Raman spectroscopy, fluorescent imaging, and square wave voltammetry, we demonstrate that thiolated DNA is concentrated and immobilized on gold surfaces with an upright conformation. Moreover, our results reveal that the freezing-induced DNA surfaces are more uniform, leading to improved DNA stability and target recognition. Lastly, we demonstrate the successful detection of a model drug in undiluted whole blood while mitigating the effects of biofouling. Our work not only provides a simple approach to tailor the DNA-gold surface for biosensors but also sheds light on the unique behavior of DNA oligonucleotides upon freezing on the liquid-solid interface.

Effect of Tween 80 on the Stability and Conformation of Telomeric G-Quadruplex DNA

G-quadruplex DNA is formed in telomer. It inhibits telomerase enzyme which is found active in more than 85 % of cancer cells. In this work, the effect of Tween 80 on telomeric G-quadruplex DNA, AGGG(TTAGGG)3 was studied using circular dichroism spectroscopic technique. The obtained results showed that using Tween 80 with telomeric quadruplex is safe up to less than 1.0 %.

BRCA1 the Versatile Defender: Molecular to Environmental Perspectives.

The evolving history of BRCA1 research demonstrates the profound interconnectedness of a single protein within the web of crucial functions in human cells. Mutations in BRCA1, a tumor suppressor gene, have been linked to heightened breast and ovarian cancer risks. However, despite decades of extensive research, the mechanisms underlying BRCA1's contribution to tissue-specific tumor development remain elusive. Nevertheless, much of the BRCA1 protein's structure, function, and interactions has been elucidated. Individual regions of BRCA1 interact with numerous proteins to play roles in ubiquitination, transcription, cell checkpoints, and DNA damage repair. At a cellular scale, these BRCA1 functions coordinate tumor suppression, R-loop prevention, and cellular differentiation, all of which may contribute to BRCA1's role in cancer tissue specificity. As research on BRCA1 and breast cancer continues to evolve, it will become increasingly evident that modern materials such as Bisphenol A should be examined for their relationship with DNA stability, cancer incidence, and chemotherapy. Overall, this review offers a comprehensive understanding of BRCA1's many roles at a molecular, cellular, organismal, and environmental scale. We hope that the knowledge gathered here highlights both the necessity of BRCA1 research and the potential for novel strategies to prevent and treat cancer in individuals carrying BRCA1 mutations.

Altered H3K4me3 profile at the TFAM promoter causes mitochondrial alterations in preadipocytes from first-degree relatives of type 2 diabetics

BackgroundFirst-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR.ResultsSequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression.ConclusionsHistone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk.Graphical abstract

TDP-43 Epigenetic Facets and Their Neurodegenerative Implications.

Since its initial involvement in numerous neurodegenerative pathologies in 2006, either as a principal actor or as a cofactor, new pathologies implicating transactive response (TAR) DNA-binding protein 43 (TDP-43) are regularly emerging also beyond the neuronal system. This reflects the fact that TDP-43 functions are particularly complex and broad in a great variety of human cells. In neurodegenerative diseases, this protein is often pathologically delocalized to the cytoplasm, where it irreversibly aggregates and is subjected to various post-translational modifications such as phosphorylation, polyubiquitination, and cleavage. Until a few years ago, the research emphasis has been focused particularly on the impacts of this aggregation and/or on its widely described role in complex RNA splicing, whether related to loss- or gain-of-function mechanisms. Interestingly, recent studies have strengthened the knowledge of TDP-43 activity at the chromatin level and its implication in the regulation of DNA transcription and stability. These discoveries have highlighted new features regarding its own transcriptional regulation and suggested additional mechanistic and disease models for the effects of TPD-43. In this review, we aim to give a comprehensive view of the potential epigenetic (de)regulations driven by (and driving) this multitask DNA/RNA-binding protein.

Fission yeast Cdc14-like phosphatase Flp1/Clp1 modulates the transcriptional response to oxidative stress

Reactive oxygen species (ROS) are an important source of cellular damage. When ROS intracellular levels increase, oxidative stress takes place affecting DNA stability and metabolic functions. To prevent these effects, stress-activated protein kinases (SAPKs) delay cell cycle progression and induce a transcriptional response that activates antioxidant mechanisms ensuring cell adaptation and survival. Fission yeast Cdc14-like phosphatase Flp1 (also known as Clp1) has a well-established role in cell cycle regulation. Moreover, Flp1 contributes to checkpoint activation during replication stress. Here, we show that Flp1 has a role in fine-tuning the cellular oxidative stress response. Phosphorylation-dependent nucleolar release of Flp1 in response to oxidative stress conditions plays a role in the cellular transcriptional response. Thus, Flp1 ablation increases the transcriptional response to oxidative stress, in both intensity and duration, upregulating both Atf1/Pcr1- and Pap1-dependent stress induced genes. Remarkably, we found that Flp1 interacts with the Atf1/Pcr1 complex with Pcr1 acting as a direct substrate. Our results provide evidence that Flp1 modulates the oxidative stress response by limiting the Atf1/Pcr1-mediated transcription.

A Comprehensive Review on the Benzimidazole Scaffold as a Potential Nucleus for Anticancer Activity

Abstract: It is common knowledge that cancer is the world's biggest cause of mortality. This has led to the ongoing introduction and validation of new cancer therapy outcomes. Benzimidazole derivatives control DNA stability and cell cycle advancement. A heterocyclic nucleus is structurally more favourable for a direct DNA connection and, thus, for the management of the DNA replication process. This review seeks to explore the usefulness of such drugs in the future facets of cancer prognosis and treatment in addition to discussing the role of benzimidazole in cancer therapy. Benzimidazole derivatives have been reported as a pertinent therapeutic strategy to modify cancer progression and malignancy in the data from 2013 to 2022. Here, we emphasize these features in several cancer types and cell lines using in vitro and in vivo methods.

Prediction of DNA Methylation based on Multi-dimensional feature encoding and double convolutional fully connected convolutional neural network.

DNA methylation takes on critical significance to the regulation of gene expression by affecting the stability of DNA and changing the structure of chromosomes. DNA methylation modification sites should be identified, which lays a solid basis for gaining more insights into their biological functions. Existing machine learning-based methods of predicting DNA methylation have not fully exploited the hidden multidimensional information in DNA gene sequences, such that the prediction accuracy of models is significantly limited. Besides, most models have been built in terms of a single methylation type. To address the above-mentioned issues, a deep learning-based method was proposed in this study for DNA methylation site prediction, termed the MEDCNN model. The MEDCNN model is capable of extracting feature information from gene sequences in three dimensions (i.e., positional information, biological information, and chemical information). Moreover, the proposed method employs a convolutional neural network model with double convolutional layers and double fully connected layers while iteratively updating the gradient descent algorithm using the cross-entropy loss function to increase the prediction accuracy of the model. Besides, the MEDCNN model can predict different types of DNA methylation sites. As indicated by the experimental results,the deep learning method based on coding from multiple dimensions outperformed single coding methods, and the MEDCNN model was highly applicable and outperformed existing models in predicting DNA methylation between different species. As revealed by the above-described findings, the MEDCNN model can be effective in predicting DNA methylation sites.

Detection of African Swine Fever Virus in Feed and Feed Mill Environment Following Extended Storage

One way to mitigate risk of feed-based pathogens for swine diets is to quarantine feed ingredients before inclusion in complete diets. Data have been generated evaluating the stability of swine viruses in ingredients, but the stability of African swine fever virus (ASFV) in feed or in a feed manufacturing environment has not been well characterized. Therefore, this study aimed to determine the stability of ASFV DNA in swine feed and on mill surfaces over time. A pilot-scale feed mill was used to manufacture six sequential batches of feed consisting of a batch of ASFV-free feed, followed by a batch inoculated with ASFV (final concentration = 5.6 × 104 TCID50/g), and then four subsequent ASFV-free batches. After each batch, 10 feed samples were aseptically collected in a double “X” pattern. During feed manufacturing, 24 steel coupons were placed on the floor of the manufacturing area and allowed to collect dust during feed manufacturing. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. Three of each were randomly selected from storage on 3, 7, 14, 28, 60, 90, and 180 days after feed manufacturing and analyzed for ASFV DNA. For feed samples, there was evidence of a batch × day interaction ( P = 0.023 ) for the quantification of genomic copies/g of feed, indicating that the amount of ASFV DNA present was impacted by both the batch of feed and days held at room temperature. There were no differences of genomic copies/g in early batches, but quantity of detectable ASFV decreased with increasing storage time. In Batches 4–6, the greatest quantity of ASFV DNA was detected on the day of feed manufacturing. The lowest quantity was detected on Day 7 for Batch 4, Day 60 for Batch 5, and at 28 and 180 days for Batch 6. There was no evidence of ASFV degradation on environmental discs across holding times ( P = 0.433 ). In conclusion, the quarantining of feed may help reduce but not eliminate the presence of ASFV DNA in feed over time. Importantly, ASFV DNA was detectable on feed manufacturing surfaces for at least 180 days with no overt evidence of reduction, highlighting the importance of bioexclusion of ASFV within feed manufacturing facilities and the need for thorough/effective decontamination and other mitigation processes in affected areas.

Interactions of ammonium based ionic liquids with DNA in aqueous medium through Volumetric, acoustic and viscometric properties

Ionic liquids (ILs) have emerged as a promising alternative solvent for DNA based studies as most of the studied ILs enhance the stability of DNA and act as a media for extraction and storage. Investigation of IL-DNA interactions through spectroscopic and computational analysis have been carried out earlier and we here attempt to explore the interaction through physicochemical properties of IL-DNA mixture in water medium that provide fundamental knowledge of the solvation behaviour. Properties such as density, ρ, speed of sound, u and viscosity, η, of two ILs, Benzyltributylammonium chloride ([BTBA]Cl) and Tetrabutylammonium bromide ([TBA]Br) in water as well as in aqueous DNA solutions with varying concentrations were measured over a temperature range from 293.15 − 323.15 K and at atmospheric pressure. The derived volumetric properties were calculated to understand the strength and extent of the solute–solvent interactions prevail in the solution. The analysis of partial molar volume at infinite dilution, V2∞, helped in the comparison of the strength of the interactions in between the studied solutes. The second derivative of V2∞ with respect to temperature, (∂2V2∞/∂T2)P facilitated in identifying the influence of the solutes in water structure formation around the solute and it was interesting to note that, although both the ILs behaved similarly in water as structure makers, [TBA]Br showed variation in the presence of DNA by demonstrating water structure breaking effects. Similar results were obtained from the analysis of viscometric properties as well. Further, the capability of the ILs to stay solvated in the DNA aqueous solution was confirmed by the results from the solvation number calculations.

Multigenerational Effects of Graphene Oxide Nanoparticles on Acheta domesticus DNA Stability.

The use of nanoparticles like graphene oxide (GO) in nanocomposite industries is growing very fast. There is a strong concern that GO can enter the environment and become nanopollutatnt. Environmental pollutants' exposure usually relates to low concentrations but may last for a long time and impact following generations. Attention should be paid to the effects of nanoparticles, especially on the DNA stability passed on to the offspring. We investigated the multigenerational effects on two strains (wild and long-lived) of house cricket intoxicated with low GO concentrations over five generations, followed by one recovery generation. Our investigation focused on oxidative stress parameters, specifically AP sites (apurinic/apyrimidinic sites) and 8-OHdG (8-hydroxy-2'-deoxyguanosine), and examined the global DNA methylation pattern. Five intoxicated generations were able to overcome the oxidative stress, showing that relatively low doses of GO have a moderate effect on the house cricket (8-OHdG and AP sites). The last recovery generation that experienced a transition from contaminated to uncontaminated food presented greater DNA damage. The pattern of DNA methylation was comparable in every generation, suggesting that other epigenetic mechanisms might be involved.

Biological interaction of Pt complex with imidazole derivative as an anticancer compound with DNA: Experimental and theoretical studies

This investigation is applied to find out interesting information on DNA binding mode with Pt(II) derivative of two N, N bidentate ligands in treating cancer. Thus, one new water-soluble platinum complex with FIP and phen with a new formula of [Pt(phen)(FIP)](NO3)2 was prepared and specified. DFT data can be used to evaluate geometry parameters. Based on the ADMET prediction, this complex can be considered a drug-like agent. Cytotoxicity property was evaluated against some human cancerous MCF7, A549, and HCT116 cell lines. Accumulation of Pt complex, cisplatin, and oxaliplatin in each cancerous cell was determined, which is probably related to their lipophilicity and solubility properties. The binding mode of the complex to ct-DNA was investigated by fluorescence spectroscopy, circular dichroism, and molecular docking simulation. The viscosity of DNA by different concentrations of EB and Pt complex titration shows Pt complex interacts with DNA via groove binding like the spectroscopic binding result. In the MD study, DNA helix, RMSD, and RMSF analysis showed that DNA stability decreased and that the majority of residues left the initial state. DNA increased residual deviations and flexibility are linked to an increase in its gyratory radius, which is consistent with the findings of the experiments.

Suitability of double-stranded DNA as a molecular standard for the validation of analytical ultracentrifugation instruments.

To address the current lack of validated molecular standards for analytical ultracentrifugation (AUC), we investigated the suitability of double-stranded DNA molecules. We compared the hydrodynamic properties of linear and circular DNA as a function of temperature. Negatively supercoiled, nicked, and linearized 333 and 339bp minicircles were studied. We quantified the hydrodynamic properties of these DNAs at five different temperatures, ranging from 4 to 37°C. To enhance the precision of our measurements, each sample was globally fitted over triplicates and five rotor speeds. The exceptional stability of DNA allowed each sample to be sedimented repeatedly over the course of several months without aggregation or degradation, and with excellent reproducibility. The sedimentation and diffusion coefficients of linearized and nicked minicircle DNA demonstrated a highly homogeneous sample, and increased with temperature, indicating a decrease in friction. The sedimentation of linearized DNA was the slowest; supercoiled DNA sedimented the fastest. With increasing temperature, the supercoiled samples shifted to slower sedimentation, but sedimented faster than nicked minicircles. These results suggest that negatively supercoiled DNA becomes less compact at higher temperatures. The supercoiled minicircles, as purified from bacteria, displayed heterogeneity. Therefore, supercoiled DNA isolated from bacteria is unsuitable as a molecular standard. Linear and nicked samples are well suited as a molecular standard for AUC and have exceptional colloidal stability in an AUC cell. Even after sixty experiments at different speeds and temperatures, measured over the course of 4months, all topological states of DNA remained colloidal, and their concentrations remained essentially unchanged.

A Solution for Ambient Storage of DNA

The need for a solution for room temperature storage of DNA is well known. Studies were conducted to assess stability of EBV DNA using Daykin Molecular Solutions GeneGuardDNA (Georgia). All laboratory testing was done at Prime Laboratories in Van Nuys California. The objective is to demonstrate sample stability of EBV DNA during shipping, storage, and multiple freeze/thaw cycles. To achieve the objectives, the study was divided into two parts; Part 1 to demonstrate stability during shipping and multiple freeze/thaw cycles. Samples were labeled as P1-1 (Positive sample 1, P1-2 first freeze/thaw, P1-3 second freeze/thaw, etc.). In Part 1 the standard deviation between positive samples ranged from 0.08 CTs to 0.25 CTs with no gradual deterioration detected with more freeze/thaw cycles). The average was 0.17 CT’s which is within the instrument’s precision range. In the past we have had no instance of repeating the same extracted sample more than two times. In Part 2 we demonstrated stability during storage. 185 archived clinical samples stored at room temperature from 2013 through 2017 were randomly chosen. DNA was extracted from these samples and EBV DNA assay testing was performed. The results from the new test were compared to the original results obtained for the samples. Concordance and p values were calculated to show the effects of long-term storage on the results of the EBV DNA assay. Pearson correlation coefficient between the original and recent assay results was 0.9564 with a p value of p<0.001 indicating statistically significant correlation between the two assay results. EBV DNA showed no deterioration of sample Ct signal was observed due to Freeze/Thaw cycles and the results were stable across multiple cycles for a period of 6 years at room temperature.