Small Amounts Of DNA Research Articles

Serological and Molecular Diagnosis of Human Cytomegalovirus among Hemodialysis Patients in Kirkuk/Iraq

Serological and Molecular Diagnosis of Human Cytomegalovirus among Hemodialysis Patients in Kirkuk/Iraq

Assessment of Genomic Integrity of Vitex negundoL., An Important Indian Medicinal Plant, Using RAPD Markers

Vitex negundo is an Indian medicinal plant containing steroids, flavonoids, lignans and terpenoids that can be used as a precursor for commercial production. An efficient marker system such as Random Amplified Polymorphic DNA (RAPD) was used to assess the genetic integrity of V. negundo. The straightforward method RAPD can be used to evaluate genomic integrity because it uses a small amount of DNA for PCR amplification. Six out of thirteen RAPD primers generated 150 distinct bands, of which 31 were polymorphic, with an average of 5.16 polymorphic bands per primer. A maximum of up to 32 fragments were amplified, and an average of 25 per primer, and the amplicons varied in size between 100 and 2000bp. The percentage of polymorphism ranges from 12.9 to 22.5, with an average of 16.6. The PIC values ranged from 0.11 to 0.63 for RAPD primers. The study pointed out that RAPD markers evaluate the genetic fidelity in Vitex negundo. The UPGMA cluster analysis grouped all in vitro raised plantlets treated with different growth regulators such as BAP, DPU, TDZ, and mT. The principal component analysis also substantiates this clustering pattern. Thus, the phylogenetic relationship and a high genetic variation revealed in the present study could provide baseline data for the conservation and improvement of this plant in future. Also, the molecular marker identified in this study will be helpful in the authentication of this species to prevent adulteration in herbal medicine.

Discovery unbalanced DNA mixtures and evaluation mixing ratio via a droplet digital PCR method.

Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator's conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures. The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios. The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately. A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

Luciferase Reporter Assay for Determining the Signaling Activity of Interferons.

The classic dual luciferase reporter assay has been widely used to rapidly and accurately determine the transcriptional activity of a given promoter induced by certain signal pathways in the cells. In particular, the sensitive characteristics of luciferase highlight its significance in many experiments, such as weak promoter analysis, transfection studies using small amounts of DNA, and detection in cell lines with low transfection efficiency. This chapter presents detailed information and experimental procedures for measuring interferon (IFN)-induced Interferon-Stimulated Response Element (ISRE) promoter activity using the dual luciferase reporter assay.

Enhanced DNA Raman modes probed by SiO2 photonic crystals

The study of large biomolecules, in particular DNA, is of practical interest. Marker bands of the Raman spectrum of DNA provide information about the conformational state and structure of the macromolecule. At the same time, when dealing with biological experiments, a small amount of DNA is available, which is why it is important to enhance the signal from DNA and find the proper substrate. However, it is difficult to enhance Raman spectra for large molecules as DNA without functionalization that could introduce changes in the DNA structure. In this work, a conventional substrate as CaF2 and a photonic crystal as a substrate with enhancer signal property and without DNA functionalization were applied. The factor of enhancement for DNA from salmon testes was reached up to 10 without Raman spectra distortion. The Raman spectral bands on different types of substrates are analyzed and compared.

What fraction of cellular DNA turnover becomes cfDNA?

Cell-free DNA (cfDNA) tests use small amounts of DNA in the bloodstream as biomarkers. While it is thought that cfDNA is largely released by dying cells, the proportion of dying cells' DNA that reaches the bloodstream is unknown. Here, we integrate estimates of cellular turnover rates to calculate the expected amount of cfDNA. By comparing this to the actual amount of cell type-specific cfDNA, we estimate the proportion of DNA reaching plasma as cfDNA. We demonstrate that &lt;10% of the DNA from dying cells is detectable in plasma, and the ratios of measured to expected cfDNA levels vary a thousand-fold among cell types, often reaching well below 0.1%. The analysis suggests that local clearance, presumably via phagocytosis, takes up most of the dying cells' DNA. Insights into the underlying mechanism may help to understand the physiological significance of cfDNA and improve the sensitivity of liquid biopsies.

What fraction of cellular DNA turnover becomes cfDNA?

Cell-free DNA (cfDNA) tests use small amounts of DNA in the bloodstream as biomarkers. While it is thought that cfDNA is largely released by dying cells, the proportion of dying cells' DNA that reaches the bloodstream is unknown. Here, we integrate estimates of cellular turnover rates to calculate the expected amount of cfDNA. By comparing this to the actual amount of cell type-specific cfDNA, we estimate the proportion of DNA reaching plasma as cfDNA. We demonstrate that <10% of the DNA from dying cells is detectable in plasma, and the ratios of measured to expected cfDNA levels vary a thousand-fold among cell types, often reaching well below 0.1%. The analysis suggests that local clearance, presumably via phagocytosis, takes up most of the dying cells' DNA. Insights into the underlying mechanism may help to understand the physiological significance of cfDNA and improve the sensitivity of liquid biopsies.

DNA Profiling in Human Identification: From Past to Present.

Forensic DNA typing has been widely accepted in the courts all over the world. This is because DNA profiling is a very powerful tool to identify individuals on the basis of their unique genetic makeup. DNA evidence is capable of not only identifying the presence of specific biospecimens in a crime scene, but it is also used to exonerate suspects who are innocent of a crime. Technological advancements in DNA profiling, including the development of validated kits and statistical methods have made this tool to be more precise in forensic investigations. Therefore, validated combined DNA index system (CODIS) short tandem repeats (STRs) kits which require very small amount of DNA, coupled with real-time polymerase chain reaction (PCR) and the statistical strengths are used routinely to identify human remains, establish paternity or to match suspected crime scene biospecimens. The road to modern DNA profiling has been long, and it has taken scientists decades of work and fine tuning to develop highly accurate testing and analyses that are used today. This review will discuss the various DNA polymorphisms and their utility in human identity testing.

B-222 Validation of a qPCR Method for Qualitative Detection of Monkeypox Virus

Abstract Background The 2022 Monkeypox virus (MPXV) outbreak became a global concern, prompting a multidisciplinary response to increase surveillance and isolate suspected and confirmed cases to reduce disease spread. Quantitative PCR (qPCR) is capable of detecting small amounts of DNA or RNA, and can be used to quickly and accurately confirm MPXV cases, even at early stages of infection, which is crucial for outbreak control and management. Methods qPCR was performed on a 384-well plate with a total reaction volume of 20 μl consisting of 9 μL Acrometrix Monkeypox DNA Positive Control at serial dilutions, 10 μL BactoPure Master Mix, and 1 μL TaqMan Monkeypox Virus Microbe Detection Assay. Positive control dilutions 103, 102, 101, and 100 were diluted with TE Buffer. qPCR was performed on two different Quantstudio 12K Flex instruments. Results The qPCR method validation was performed by calculating precision, accuracy, and linearity on serial dilutions of Acrometrix Monkeypox Virus DNA Positive Control. Precision testing was performed on 2 levels of control: low (101) and high (103) over 5 days with 4 repeats per day. Accuracy and linearity testing were performed on 4 levels with 3 repeats. Coefficient of variation (CV) was less than 5%, specifically 0.9% for the low control and 4.3% for the high control, indicating an acceptable level of precision. Percent recovery fell close to 100%, specifically within 101%-103% for all controls, indicating the results fell close to the expected value. Linearity R2 was close to 1, specifically 0.984, demonstrating the results are proportional to the concentration analyzed. Conclusion Validation of MPXV detection through qPCR was successful. Development and validation of qPCR assays for the detection of positive MPXV cases is essential for managing future outbreaks and combatting future pathogenic strains.

BO-DNA: Biologically optimized encoding model for a highly-reliable DNA data storage

DNA data storage is a promising technology that utilizes computer simulation, and synthetic biology, offering high-density and reliable digital information storage. It is challenging to store massive data in a small amount of DNA without losing the original data since nonspecific hybridization errors occur frequently and severely affect the reliability of stored data. This study proposes a novel biologically optimized encoding model for DNA data storage (BO-DNA) to overcome the reliability problem. BO-DNA model is developed by a new rule-based mapping method to avoid data drop during the transcoding of binary data to premier nucleotides. A customized optimization algorithm based on a tent chaotic map is applied to maximize the lower bounds that help to minimize the nonspecific hybridization errors. The robustness of BO-DNA is computed by four bio-constraints to confirm the reliability of newly generated DNA sequences. Experimentally, different medical images are encoded and decoded successfully with 12%–59% improved lower bounds and optimally constrained-based DNA sequences reported with 1.77bit/nt average density. BO-DNA's results demonstrate substantial advantages in constructing reliable DNA data storage.

Evaluation of the pooled sample method in Infinium MethylationEPIC BeadChip array by comparison with individual samples

BackgroundThe pooled sample method is used in epigenomic research and expression analysis and is a cost-effective screening approach for small amounts of DNA. Evaluation of the pooled sample method in epigenomic studies is performed using the Illumina Infinium Methylation 450K BeadChip array; however, subsequent reports on the updated 850K array are lacking. A previous study demonstrated that the methylation levels obtained from individual samples were accurately replicated using pooled samples but did not address epigenome-wide association study (EWAS) statistics. The DNA quantification method, which is important for the homogeneous mixing of DNA in the pooled sample method, has since become fluorescence-based, and additional factors need to be considered including the resolution of batch effects of microarray chips and the heterogeneity of the cellular proportions from which the DNA samples are derived. In this study, four pooled samples were created from 44 individual samples, and EWAS statistics for differentially methylated positions (DMPs) and regions (DMRs) were conducted for individual samples and compared with the statistics obtained from the pooled samples.ResultsThe methylation levels could be reproduced fairly well in the pooled samples. This was the case for the entire dataset and when limited to the top 100 CpG sites, consistent with a previous study using the 450K BeadChip array. However, the statistical results of the EWAS for the DMP by individual samples were not replicated in pooled samples. Qualitative analyses highlighting methylation within an arbitrary candidate gene were replicable. Focusing on chr 20, the statistical results of EWAS for DMR from individual samples showed replicability in the pooled samples as long as they were limited to regions with a sufficient effect size.ConclusionsThe pooled sample method replicated the methylation values well and can be used for EWAS in DMR. This method is sample amount-effective and cost-effective and can be utilized for screening by carefully understanding the effective features and disadvantages of the pooled sample method and combining it with candidate gene analyses.

PGT-A: Houston, we have a problem.

Preimplantation genetic testing for aneuploidy (PGT-A) is a common add-on to IVF cycles. As it is presently performed, PGT-A relies on whole genome amplification of small amounts of DNA from cells removed from the trophectoderm (TE) of a blastocyst for determination of gain or loss of chromosomal material by next-generation sequencing. Whole genome amplification may introduce artifacts such as allele dropout and loss of heterozygosity in up to 25% of cases. In addition, the high prevalence of mosaicism in human embryos is a complicating factor in interpreting the results of PGT-A screening. In the presence of mosaicism, biopsy of TE cells cannot provide accurate results regarding the chromosomal make-up of the inner cell mass. The available clinical data suggest that PGT-A is probably harmful when IVF outcomes are analyzed by intention to treat or by live birth rate per cycle started rather than per embryo transfer, especially in women with three or fewer blastocysts. In addition, hypothesized advantages of reduced spontaneous abortion rate and reduced time to conception may be modest at best.

Detection of Epstein–Barr virus infection in thymic epithelial tumors by nested PCR and Epstein–Barr-encoded RNA ISH

BackgroundEpstein–Barr virus (EBV) is well known to be associated with a lot of tumors, including lymphoma, nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and some other carcinomas with similar lymphoepithelioma-like features. However, the association between EBV and thymic epithelial tumors (TETs) is inconclusive as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. The geographical difference of the patients is also one of the reasons for the different points of view.MethodsIn our study, we examined 72 thymomas, including 3 cases of type A thymomas, 27 cases of type AB, 6 cases of type B1, 26 cases of type B2 and 10 cases of type B3 thymomas, and 15 thymic carcinomas to detect the viral genome at both DNA and RNA levels. The genome DNA of fresh tissues was first screened by nested polymerase chain reaction (PCR), which could be regarded as the most sensitive method to detect small amounts of DNA. Then all the tissue blocks were further submitted for viral localization by Epstein–Barr-encoded RNA (EBER) ISH. Group parameters were assessed using the chi-square test at a significance level of p < 0.05.ResultsNested PCR results showed that none of type A, eight (29.6%) type AB, one (16.7%) type B1, fifteen (57.7%) type B2, and four (40.0%) type B3 were positive for EBV genome. However, none of them detected EBER expression except for one case of type B2 thymoma. Fourteen (93.3%) thymic carcinomas were positive for EBV by nested PCR, of which three displayed weak nuclear signals within the tumor cells by EBER ISH.ConclusionsThese results showed that nested PCR was a sensitive method for screening the EBV genome in thymic epithelial tumors. As the malignancy of thymoma increases, the rate of EBV infection became higher. Thymic carcinomas were well associated with the Epstein–Barr virus.There was significant association between the EBV infection rate and thymoma type (p < 0.05). We further analyzed the association between EBV infection and myasthenia gravis. However, it showed no significant difference(p = 0.2754), although the EBV infection rate was higher in the thymomas with myasthenia gravis.

An animal-friendly collection of samples from the upper respiratory tract via drinker-swabs for the detection of avian influenza (H5N1) in poultry flocks

To take tracheal swabs from poultry flocks, for detection of avian influenza viruses is time-consuming and involves a high workload for the staff and a lot of stress for the sampled animals. Therefore, an animal-friendly and at the same time reliable method of collected of samples from poultry flocks is needed. A simple method would be a significant improvement in the control of avian influenza, especially in poultry-intensive regions. Swab samples in/on drinkers can easily be taken. When taking water, every animal with a respiratory infection leaves small amounts of its respiratory secretions (mucus) in/on the drinkers. Even before the first clinical signs appear in a flock, the influenza virus is already in small amounts of mucus particle in the drinkers present. By sampling swabs in/on many drinkers in the barn these smallest amounts of respiratory murus from infected birds in the flock can be detected by PCR-technic. In contrast to the faecal samples, the samples from the drinkers contain only a small amount of DNA and RNA from microorganisms&#x0D; The PCR results from this field study allow the conclusion that swabs from the drinkers in the barn yield very reliable and very important information about the presence of avian influenza viruses in a poultry population. However, it is important to take swab samples from many drinkers in the barn. An Influenza monitoring with swab samples from drinkers is rapid, sensitive, specific, reliable, inexpensive and can be taken easily by individuals, regardless of the age and the number of birds in the flock. Detecting the avian influenza virus in infected flocks with swab sampling in/on drinkers is an animal-friendly process with an improvement in the statistical significance of the infection status of a flock.

Morphological to Molecular Markers: Plant Genetic Diversity Studies in Walnut (Juglans regia L.)—A Review

Persian walnut populations have tremendous morphological and allelic diversity in their germplasm due to heavy outcrossing and years of seed multiplication. These variations are assessed by morphological, cytological, biochemical, and molecular markers. Various researchers have used different tree, foliage, flower, nut, and kernel traits to evaluate morphological/phenotypic diversity. In walnut, morphological indices are first taken into consideration to describe and classify the germplasm, but the environment influences them. In comparison, DNA-based markers can detect genetic diversity at any stage of plant development and have been shown to be a potential tool for assessing variation at the DNA level and deciphering genetic relationships within and between species. Microsatellites are very powerful and informative among DNA-based markers in studying genetic relationships and genetic identity at different levels. They are neutral, highly frequent, uniformly distributed, hypervariable, codominant, highly reproducible, produce many alleles per locus, and require a small amount of DNA for analysis. Current breeding objectives can be achieved by selecting superior genotypes from the germplasm, supplemented by molecular characterization in the selection of parents for each breeding program. Therefore, the use of morphological and molecular markers is recommended for efficient exploration and utilization of germplasm resources and to improve diversity among genetic resources. The published literature on morphological and molecular markers, especially simple sequence repeats (SSRs), is presented in this review to provide current insights into the level of genetic diversity in walnut.

Optimized Workflow for Whole Genome and Transcriptome Next‐Generation Sequencing of Single Cells or Limited Nucleic Acid Samples

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.

Applying Unique Molecular Indices with an Extensive All-in-One Forensic SNP Panel for Improved Genotype Accuracy and Sensitivity

One of the major challenges in forensic genetics is being able to detect very small amounts of DNA. Massively parallel sequencing (MPS) enables sensitive detection; however, genotype errors may exist and could interfere with the interpretation. Common errors in MPS-based analysis are often induced during PCR or sequencing. Unique molecular indices (UMIs) are short random nucleotide sequences ligated to each template molecule prior to amplification. Applying UMIs can improve the limit of detection by enabling accurate counting of initial template molecules and removal of erroneous data. In this study, we applied the FORCE panel, which includes ~5500 SNPs, with a QIAseq Targeted DNA Custom Panel (Qiagen), including UMIs. Our main objective was to investigate whether UMIs can enhance the sensitivity and accuracy of forensic genotyping and to evaluate the overall assay performance. We analyzed the data both with and without the UMI information, and the results showed that both genotype accuracy and sensitivity were improved when applying UMIs. The results showed very high genotype accuracies (>99%) for both reference DNA and challenging samples, down to 125 pg. To conclude, we show successful assay performance for several forensic applications and improvements in forensic genotyping when applying UMIs.

BIOLOGICAL SAMPLES CONTAMINATION CONTROL OF THE SUS SCROFA USING HAPLOID DNA MARKERS

This paper proposes an effective method for controlling the contamination of biological samples of Sus scrofa with alien material in the preanalytical phase of a PCR study. Because PCR is highly sensitive, even a small amount of DNA containing alien biological substances can lead to false results. In the case of analysis of contaminated biological samples using diploid DNA markers, a mixture of two different homozygotes will be defined as a heterozygote. Unlike diploid DNA markers, a mixture of two different haplotypes is uniquely determined. To perform the study in the slaughter shop of the Globinsky Meat Processing Plant, after slaughter, one ear was cut off from the carcasses of pigs with an animal identification number tag. DNA was isolated from the epithelial tissue of the auricle. Five SNPs of the mitochondrial genome were used as a haploid marker. The study was carried out using a multisite PCR-RFLP method, the peculiarity of which was the analysis of the D-loop fragment between positions 15531 and 15959 of the porcine mitochondrial genome (GenBank: AJ002189.1). This sequence contains one monomorphic site (15558W) and five polymorphic Tas I restriction enzyme sites (15616T &gt; C, 15714T &gt; C, 15758T &gt; C, and 15916A &gt; T). The presence or absence of the Tas I site in the above positions determines mitochondrial haplotypes, denoted by Latin letters from A to P. PCR-RFLP analysis of DNA samples revealed DNA fragments on the electrophoregram, indicating a mixture of two or more haplotypes. It was possible to establish the presence of contamination due to the use of a multisite PCR-RFLP method, which implies a strictly discrete set of restrictive fragments for the mitochondrial DNA of an individual animal. The total size of the restriction fragments should be 428 bp and the presence of additional DNA bands indicate the association of two or more haplotypes. Thus, it was demonstrated that the use of haploid DNA markers makes it possible to determine the contamination of samples with alien material. This method can be used in the study of porcine nuclear DNA as a laboratory quality assurance of the preanalytical phase, which will reduce laboratory costs, improve the organization of work and avoid dramatic errors when performing genetic examinations. Keywords: pigs, epithelium, mitochondrial genome, haploid DNA markers, PCR, contamination.

Improvement of Bacterial Transformation Efficiency Using Biopolymers as Plasmid Complements

The process of bacterial transformation results from the introduction of specific genetic material into bacteria cells and subsequent gene expression. This technology has significant applications in gene cloning technology, and allows new possibilities for genetic breeding. The use of cellulose and babassu mesocarp to improve the process was not found in the literature. Thus, the objective of this work was to evaluate the effect of biopolymers (cellulose and biopolymer rich in starch extracted from the babassu mesocarp) on the efficiency of this process. The biopolymers were characterized by XRD, FTIR, zeta potential, thermal analysis, agarose gel electrophoresis and DLS. The transformation was carried out using the plasmid pGEM and E. coli. It was investigated the cytotoxicity and the effect of different concentrations of the materials and plasmid DNA on the transformation. The study demonstrated that 0.050 mg/mL of both materials increased the efficiency of bacteria transformation, and small amount of DNA combining with the biopolymers allowed obtaining satisfactory results. Therefore, the biopolymers can be used in genetic studies involving the transfer of DNA into bacterial cells.

RTX 훈증 기법을 이용한 담배에 형성된 잠재지문의 현출

Cigarette-related garbage can be easily found on anywhere, even at the crime scene. Cigarette evidence can provide DNA evidence and have been used in investigation for a long time. If very small amounts of DNA are detected or in a mixture with contaminants, it is hard to analysis DNA In this case, fingerprints can be conclusive evidence to identify suspects. However, when 1,2-indanedione (1,2-IND) was applied to cigarettes, the background was stained and made it difficult to identify ridges. Therefore, latent fingerprint development research is needed in such case, and when DNA analysis is difficult on cigarettes. In this study, 1,2-IND and Ruthenium tetroxide (RTX) were used to compare the development of latent fingerprints on cigarettes, which are porous surfaces. On the filter part of cigarettes, latent fingerprints were clearly developed on both 1,2-IND and RTX, but on the body part of cigarettes, 1,2-IND reacted with tobacco leaves and showed a fluorescence reaction on the background, making it difficult to identify the ridges of the fingerprints. On the other hand, when RTX was applied, clear ridges were appeared on the body part of cigarettes. In addition, the RTX did not leave many stains on the background. The 1,2-IND is currently the most effective latent fingerprint development technique on porous surfaces but it is estimated to react with amino acid components contained in a cigarette. Therefore, when there is a need to develop latent fingerprints on cigarettes, it is confirmed that RTX reagent is much more effective than 1,2-IND reagent.