DNA Contamination Research Articles

Implementation of <i>in-house</i> Methods for Isolating Fungal DNA of Clinical Samples

Over 200 species of fungi are responsible for a variety of infections that can occur in various parts of the human body. There are several phenotypic methods for identifying these fungal elements; however, these approaches have limitations. Molecular methods are now routinely used in well-equipped mycology laboratories. However, the first step in isolating genetic material can often be costly, can suffer from external DNA contamination and some components have toxicity for personnel. The general objective of this work was to identify the best local method for isolating genetic material from fungi based on cost, yield and time-consuming criteria. A total of ten (10) different nucleic acid isolation methods were tested. Those tests using thermal or mechanical shock for cell lysis delivered better quality than those using chemical lysis. Thus, based on our criteria, the best methods for nucleic acid isolation and purification of fungal elements were cetyltrimethylammonium bromide (CTAB) combined with sterilized sea sand (less expensive) and the chelator Chelex® coupled with glass beads (faster).

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30µm). To reduce the supernatant volume and process time, the clarified sample was concentrated by ultrafiltration either using tangential flow filtration or centrifugal-based filtration units, depending on the process scale. The final purification step at laboratory-scale, was carried out by density gradient ultracentrifugation, the recovery of which was compared with chromatographic purification at large-scale. The virus preparations were analyzed using dynamic light scattering to verify the virus size and transmission electron microscopy for purity and virus morphology. Density gradient ultracentrifugation allowed the recovery of≥80% infectious particles and reduced the contaminant DNA and host cell proteins relatively to standard ultracentrifugation pelleting using a sucrose cushion. At large-scale, weak and strong anion-exchangers were tested and compared. The best columns allowed infectious virus recoveries as high as 77% and eliminated 92% of host cell proteins.

Comparison of mosquito and fly derived DNA as a tool for sampling vertebrate biodiversity in suburban forests in Berlin, Germany

AbstractThe use of invertebrate‐derived DNA (iDNA) is a promising non‐invasive tool to monitor wildlife. While most studies have been carried out in dense tropical and sub‐tropical forests and have focused on the use of a single category of invertebrates, this study compares the use of flies and mosquitoes‐derived DNA to assess vertebrate diversity in semi‐urban environments. We conducted our sampling in four different forest plots in Berlin, Germany. Pools of flies and non‐bloodfed mosquitoes were metabarcoded using 108‐bp vertebrate‐specific 12 S rRNA (12 S‐V5) and 94‐bp mammal‐specific 16 S rRNA (16Smam) mitochondrial markers, and individual bloodfed mosquitoes were sequenced using the 340‐bp vertebrate‐specific 12 S rRNA fragment (Mam‐12 S‐340). Most sequencing was only successful for mammal species. From the fly pools, we detected 10 mammal species using 16Smam, and six species using 12 S‐V5. From the non‐bloodfed mosquito pools, we only amplified putative contaminant DNA, indicating that mosquito females without visual signs of a blood meal carry no traces of vertebrate DNA. Finally, in the bloodfed mosquitoes, we identified four mammal species. We did not find significant differences in the proportion of mammal species detected regarding the total available number of species between sampling localities. Fly samples were easier to obtain and more abundant over the sampled localities compared to mosquito samples. We conclude that, while there are a few advantages in using mosquito blood meals, the use of flies in the detection of wildlife in a suburban environment is more effective in terms of collection of samples and detection of vertebrates, although this technique is limited to few mammal species in the urban environment.

A simple and efficient method for Taq DNA polymerase purification based on heat denaturation and affinity chromatography

Taq DNA polymerase from Thermus aquaticus(Taq) is a widely used enzyme in molecular biology research and clinical diagnostics because of its utility in DNA amplification and sequencing assays. For laboratory large-scale production of enzymes, effort, time and cost are crucial factors. Here we describe a simple and efficient method for the production of high-quality recombinant Taq DNA polymerase using a combined method based on heat denaturation and nickel affinity chromatography. His-tagged Taq DNA polymerase was overexpressed in Escherichia coli and isolated in a first step by heat denaturation of the bacterial lysate, since Taq DNA polymerase is a thermo-resistant protein, it remains in the soluble fraction with a few bacterial protein contaminants. The isolated His-tagged Taq DNA polymerase was further purified by nickel-NTA chromatography leading to a high quality and purity enzyme with a comparable activity to most of commercially available counterparts. DNA-contaminated Taq DNA polymerases constitute a major concern especially during DNA amplification because they can lead to generation of non-specific products. Since Taq DNA polymerase is a DNA binding protein, it carries during purification process bacterial DNA contaminants. We treated the purified Taq DNA polymerase with DNase to end up with a high-quality DNA-free Taq DNA polymerase.

Carbon-defect-driven persulfate activation for highly efficient degradation of extracellular DNA contaminant: Radical oxidation and electron transfer pathways

Extracellular DNA (eDNA), as a dynamic repository for antibiotic-resistant genes (ARGs), is a rising threat to public health. This work used a ball-milling method to enhance defect structures of activated carbon, and carbon defects exhibited an excellent capacity in persulfate (PS) activation for model eDNA and real ARGs degradation. The eDNA removal by defect-rich carbon with PS was 2.3-fold higher than that by unmilled activated carbon. The quenching experiment, electrochemical analysis and thermodynamic calculation showed that carbon defects could not only enhance the generation of SO4•– and •OH, but formed an electron transfer bridge between eDNA and PS, leading to the non-radical oxidation of eDNA. According to molecular calculations, the nitrogenous bases of DNA were the easiest sites to be oxidized by electron transfer pathway. This research offers a new way using defective carbon materials as PS activator for eDNA pollutants, and an insight into the non-radical mechanism of eDNA degradation.

Optimization of RNA Extraction Protocol for Rat Skeletal Muscle Samples

Aims: The present study aimed to stablish and characterize an optimized protocol conformation to obtain adequate RNA quality from rodents skeletal muscle samples for sequencing studies.
 Place and Duration of Study: The in vivo experiments and analyses were performed in the Laboratory of Biochemistry and Gene Expression – LABIEX of the Superior Institute of Biomedical Science – ISCB from the State University of Ceará - UECE. Between 2017-2020.
 Methodology: Were used 23 samples from male Wistar rat skeletal muscle, specifically from soleus muscle. Total RNA extraction was performed using the classic TRIzol® method and commercial kit, merging steps from both. Capillary electrophoresis in the Bioanalyzer platform was used for RNA quality evaluation.
 Results: (C) Analyzes of adapted protocol RNA concentration, RIN and rate 28S/18S showed satisfactory results. 28S/18S Ribosomal bands appear well defined, without small traces, which indicates RNA with high integrity and without contamination of genomic DNA.
 Conclusion: Obtained RNA quality and integrity data satisfied the exigencies for posterior RNA-seq.

Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells.

Direct analysis of mtDNA using PCR-free methods is limited by the presence of persistent, contaminating nucleic acids originating from the nuclear genome, even following stringent mitochondrial isolations. Here we describe a method developed in our laboratory that couples existing, commercially available mtDNA isolation protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). This protocol produces highly enriched mtDNA extracts from small-scale cell culture, with near-undetectable nuclear DNA contamination.

TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice.

Members of PARP family are responsible for poly(ADP-ribose) (pADPr) posttranslational modification synthesis. They are intensively studied proteins with more than 20,500 related papers in PubMed database search to date. PARG, the main enzyme that degrades pADPr, is unfairly attracted less attention, and 40 times less papers (a little more than 500) are related to its functioning. The difficulties to work with PARG knockout animals due to its early embryo lethality could be one reason for this huge difference. Mice PARG-specific antibodies are not available from any vendor, which also complicates the research process. There is one available for public PARG knockout mice line generated by KOMP project. It has LacZ cassette, which replaces three critical exons in PARG gene. Here, we present the method to genotype these mice with Taqman qPCR multiplex approach. It allows to work with a small amount of DNA material like early embryo stages and to separate maternal DNA contamination. The modification of this method is also applicable for studying PARG conditional knockouts and identifying the success of floxed PARG gene exon deletion by Cre-driven recombination.

High resolution UV spectral imaging and bio-detection with magnetic dipole quasi-BIC resonant dielectric metasurfaces

In this paper, we designed a ring-shaped metasurface based on diamond material that response in ultraviolet band, and with which the BIC mode is obtained by breaking the symmetry of its structural units. We show that only when the radiation rate of the magnetic dipole resonance of the BIC mode is equal to the dissipation loss brought by the surface covering material, the interaction between light and matter is maximally enhanced. The absorptivity of the film material is enhanced by an order of magnitude. The simulation results show that there is a linear relationship between the resonant positions of the structure and the scaling factor S varying structure sizes. When the metasurface is covered with a single DNA film, changes differently due to the absorption of the DNA. The reflection information of the system is further converted into ultraviolet absorption of DNA. Then the multiple absorption peaks form the absorption spectrum of DNA molecules. By Arranging the different scaling factor metasurface pixels on a same substrate we can obtain a specific absorption image. The absorptivity of DNA leads to the difference in brightness and darkness of the metasurface pixels, which can be processed into gray-scale images that give out the specific fingerprint information of the DNA. So, we realized calibration and analysis of DNA without spectroscopic measurements in the ultraviolet band now. The metasurface device can effectively detect the contamination of DNA by other biomolecules such as RNA. This work has great significance in promoting the application of metasurfaces in the field of biological detection and demonstrates that a specially designed metasurface can be applied in nucleic acid detection.

Easily-controllable, helper phage-free single-stranded phagemid production system

BackgroundSingle-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, the yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized.ResultsIn this report, we describe a new arabinose-inducible ss phagemid production method without helper phage infection. The newly exploited DNA derived from VCSM13 expresses the pII protein, which initiates ss DNA synthesis, under the control of the araBAD promoter. In addition, the packaging signal is deleted in the DNA to reduce the contamination of the phage-derived ss DNA. The phagemid DNA of interest, carrying the M13 origin of replication and the packaging signal, was introduced into bacterial cells maintaining the modified VCSM13 DNA as a plasmid, and the ss phagemid DNA production was induced by arabinose. The DNA recovered from the phage particles had less contamination from VCSM13 DNA, as compared to the conventional method. Moreover, we extended the method to purify the ss DNAs by using an anion-exchange column, to avoid the use of hazardous chemicals.ConclusionUsing this combination of methods, large quantities of phagemid ss DNAs of interest can be consistently obtained.

Tricky TRIC: A replication study using trophoblast retrieval and isolation from the cervix to study genetic birth defects.

Noninvasive Prenatal Diagnosis has recently been introduced for a limited number of monogenetic disorders. However, the majority of DNA diagnostics still require fetal material obtained using an invasive test. Recently, a novel technique, TRIC (Trophoblast Retrieval and Isolation from the Cervix), has been described, which collects fetal trophoblast cells by endocervical sampling. Since this technique has not been successfully replicated by other groups, we aimed to achieve this in the current study. Pregnant women referred for transvaginal chorionic villous sampling (CVS) were asked for an endocervical sample prior to CVS. The TRIC samples were processed to isolate trophoblast DNA. TRIC DNA was used in ForenSeq to determine the amount of maternal DNA contamination, and for Sanger sequencing in case of a monogenic disorder. 23%-44% of samples had a sufficiently high fetal DNA fraction to allow genetic testing, as calculated by Sanger sequencing and ForenSeq, respectively. We have been able to successfully replicate the TRIC protocol, although with a much lower success rate as described by the original study performing TRIC. As we obtained the samples in the actual clinical setting envisioned, the method in its current setup is not advisable for use in prenatal diagnostics.

An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA-Protein Cross-Links from Mammalian Cells.

DNA-Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.

Taq man multiplex real-time pcr for identification of porcine and canine dna in halal food

The efficient and timely detection of prohibited animals in Halal food is very important. This study aimed to establish a rapid detection method for porcine and canine DNA in a food product. Porcine and Canine DNA were Contaminated in halal food products using multiplex Real-Time PCR shown positive specificity only in Porcine and Canine meat but shown negative results in chicken, cow, and fish. The results from porcine showed that the lowest level is at 0.001% and 0.1% in raw meat and cooked meat heated at 120 degrees celsius for 15 min, and 0.1% in diluted canine DNA respectively. The method for detection of the contamination of porcine and canine DNA in Halal food products with multiplex real-time PCR technique is suitable and high sensitivity for detecting the DNA of prohibited animals in meat products and raw materials, such as detecting contaminated porcine DNA in sweet jelly and soup cubes.

137. Estimation of familial DNA contamination using Mendelian inconsistencies from next generation sequencing of trios

137. Estimation of familial DNA contamination using Mendelian inconsistencies from next generation sequencing of trios

Estimation of inherent bacterial DNA contamination in a qPCR master mix: concerns about background DNA of reagents

article: Estimation of inherent bacterial DNA contamination in a qPCR master mix: concerns about background DNA of reagents - Minerva Biotechnology and Biomolecular Research 2022 December;34(4):180-6 - Minerva Medica - Journals

Estimation of intrafamilial DNA contamination in family trio genome sequencing using deviation from Mendelian inheritance.

With the increasing number of sequencing projects involving families, quality control tools optimized for family genome sequencing are needed. However, accurately quantifying contamination in a DNA mixture is particularly difficult when genetically related family members are the sources. We developed TrioMix, a maximum likelihood estimation (MLE) framework based on Mendel's law of inheritance, to quantify DNA mixture between family members in genome sequencing data of parent-offspring trios. TrioMix can accurately deconvolute any intrafamilial DNA contamination, including parent-offspring, sibling-sibling, parent-parent, and even multiple familial sources. In addition, TrioMix can be applied to detect genomic abnormalities that deviate from Mendelian inheritance patterns, such as uniparental disomy (UPD) and chimerism. A genome-wide depth and variant allele frequency plot generated by TrioMix facilitates tracing the origin of Mendelian inheritance deviations. We showed that TrioMix could accurately deconvolute genomes in both simulated and real data sets.

Ancient DNA from speleothems: opportunity or challenge?

AbstractAncient environmental DNA retrieved from sedimentary records (sedaDNA) can complement fossil-morphological approaches for characterizing Quaternary biodiversity changes. PCR-based DNA metabarcoding is so far the most widely used method in environmental DNA studies, including sedaDNA. However, degradation of ancient DNA and potential contamination, together with the PCR amplification drawbacks, have to be carefully considered. Here we tested this approach on speleothems from an Alpine cave that, according to a previous palynomorphological study, have shown to contain abundant pollen grains. This offers a unique opportunity for comparing the two methods and, indirectly, trying to validate DNA-based results. The plant taxa identified by sedaDNA are fewer than those by pollen analysis, and success rate of PCR replicates is low. Despite extensive work performed following best practice for sedaDNA, our results are suboptimal and accompanied by a non-negligible uncertainty. Our preliminary data seem to indicate that paleoenvironmental DNA may be isolated from speleothems, but the intrinsic weakness of PCR-based metabarcoding poses a challenge to its exploitation. We suggest that newly developed methods such as hybridization capture, being free from PCR drawbacks and offering the opportunity to directly assess aDNA authenticity, may overcome these limitations, allowing a proper exploitation of speleothems as biological archives.

Concordance of PGT for aneuploidies between blastocyst biopsies and spent blastocyst culture medium

Research questionNon-invasive preimplantation genetic testing for aneuploidies (niPGT-A) avoids the possible detrimental impact of invasive PGT-A on embryo development and clinical outcomes. Does cell-free DNA (cfDNA) from spent blastocyst culture medium (BCM) reflect embryonic chromosome status better than trophectoderm (TE) biopsy? DesignIn this study, 35 donated embryos were used for research and the BCM, TE biopsy, inner cell mass (ICM) and residual blastocyst (RB) were individually picked up from these embryos. Whole genome amplification (WGA) was performed and amplified DNA was subject to next-generation sequencing. Chromosome status concordance was compared among the groups of samples. ResultsThe WGA success rates were 97.0% (TE biopsy), 100% (ICM), 97.0% (RB) and 88.6% (BCM). Using ICM as the gold standard, the chromosomal ploidy concordance rates for BCM, TE biopsy and RB were 58.33% (14/24), 68.75% (22/32) and 78.57% (22/28); the diagnostic concordance rates were 83.33% (20/24), 87.50% (28/32) and 92.86% (26/28); and the sex concordance rates were 92.31% (24/26), 100% (32/32) and 100% (28/28), respectively. Considering RB the gold standard, the chromosome ploidy concordance rates for BCM and TE biopsy were 61.90% (13/21) and 81.48% (22/27); the diagnostic concordance rates were 71.43% (15/21) and 88.89% (24/27); and the sex concordance rates were 91.30% (21/23) and 100% (27/27), respectively. ConclusionsThe results of niPGT-A of cfDNA of spent BCM are comparable to those of invasive PGT-A of TE biopsies. Modifications of embryo culture conditions and testing methods will help reduce maternal DNA contamination and improve the reliability of niPGT-A.

Toward efficient and high-fidelity metagenomic data from sub-nanogram DNA: evaluation of library preparation and decontamination methods

BackgroundShotgun metagenomic sequencing has greatly expanded the understanding of microbial communities in various biological niches. However, it is still challenging to efficiently convert sub-nanogram DNA to high-quality metagenomic libraries and obtain high-fidelity data, hindering the exploration of niches with low microbial biomass.ResultsTo cope with this challenge comprehensively, we evaluated the performance of various library preparation methods on 0.5 pg–5 ng synthetic microbial community DNA, characterized contaminants, and further applied different in silico decontamination methods. First, we discovered that whole genome amplification prior to library construction led to worse outcomes than preparing libraries directly. Among different non-WGA-based library preparation methods, we found the endonuclease-based method being generally good for different amounts of template and the tagmentation-based method showing specific advantages with 0.5 pg template, based on evaluation metrics including fidelity, proportion of designated reads, and reproducibility. The load of contaminating DNA introduced by library preparation varied from 0.01 to 15.59 pg for different kits and accounted for 0.05 to 45.97% of total reads. A considerable fraction of the contaminating reads were mapped to human commensal and pathogenic microbes, thus potentially leading to erroneous conclusions in human microbiome studies. Furthermore, the best performing in silico decontamination method in our evaluation, Decontam-either, was capable of recovering the real microbial community from libraries where contaminants accounted for less than 10% of total reads, but not from libraries with heavy and highly varied contaminants.ConclusionsThis study demonstrates that high-quality metagenomic data can be obtained from samples with sub-nanogram microbial DNA by combining appropriate library preparation and in silico decontamination methods and provides a general reference for method selection for samples with varying microbial biomass.

Analysis of an ancestry using cremated old human remains from the Korean War victims

Analysis of DNA from burnt bone fragment is the very hard work for the human identification in forensic casework. In general, cremated bone with an artificial damage is more difficult to get an intact DNA than a singed one because of the chemical and biological drastic changes such as protein denaturation and destruction. In this study, we pursue the best technical approach for the minimal damage and the contamination of DNA from other factors in the preconditioning and the extraction process based on over 70 years old Korean War victim skeletal that was burnt and buried in Korean Peninsula. First of all, we removed the pollutant and the dust from the burnt bones using dental instruments, and then incubated with EDTA buffer at 25 ℃ to remove inhibitors such as calcium and mineral. In order to compare the DNA preservation ability between a pellet and a supernatant, samples are repeatedly tested to collect washed EDTA buffer several times to separate. Each of isolated materials is secondly cleaned with the organic extraction method using phenol and analyzed mtDNA sequence with the in-house method for the ancestry assay. The better discrimination ability was appeared in the supernatant than the pellet. Nevertheless, many of the forensic geneticists use a powdering method for getting more DNA, we applied EDTA buffer in the preconditioning step to eliminate every contamination. As a result, the contamination factor was efficiently removed and the ancestry was estimated as per the written information. Consequently, cremated bone is identified to belong in the D4 mtDNA haplogroup which is commonly reported in ethnic groups in Asia especially Korea. This is a preliminary study of a human identification over an ancestry analysis to give information against a mass disaster in a future. Through a higher process optimization and better analytical methods toward more remains, which are genetically difficult to analyze, will support to examine the identity of the post cremated remains.