Presence Of Background DNA Research Articles

Where did it go? A study of DNA transfer in a social setting

The sensitivity of DNA analysis has progressed to the point that trace levels of DNA, originating from only a few cells, can generate informative profiles. This means that virtually any item or surface can be sampled with a reasonable chance of obtaining a DNA profile. As the presence of DNA does not suggest how it was deposited, questions are often raised as to how the DNA came to be at a particular location and the activity that led to its deposition. Therefore, understanding different modes of DNA deposition, reflective of realistic forensic casework situations, is critical for proper evaluation of DNA results in court. This study aimed to follow the movements of DNA to and from individuals and common household surfaces in a residential premises, while socially interacting. This took place over an hour and involved four participants, with known shedder status, designated as visitors (a male and a female) and hosts (a male and a female), who engaged in the activity of playing a board game while being served food. During the study, the participants were instructed to use the toilet on a single occasion to assess the transfer of DNA to new and unused underwear that was provided. All contacts made by the participants in the dining room and kitchen were video recorded to follow the movements of DNA. Samples were collected based on the history of contact, which included hands, fingernails and penile swabs. Direct contacts resulted in detectable transfer (LR > 1) in 87 % (87/100) of the non-intimate samples and clothing. For surfaces touched by multiple participants, DNA from the person who made the last contact was not always detectable. The duration and number of contacts did not significantly affect the detection of the person contacting the item. On the other hand, presence of background DNA and participant’s shedder status appear to play an important role. Further, unknown contributors were detected in the majority of samples. Finally, indirect transfer was observed on a number of occasions including co-habiting partners of guests who were not present at the study location. The results of this study may assist with decision making for exhibit selection or targeting areas for sampling within the home environment. Our findings can also be used in conjunction with previous literature to develop activity-level evaluations in such situations where the source of the DNA is conceded, but the mode of deposition is disputed.

Trace DNA Transfer in Co-Working Spaces: The Importance of Background DNA Analysis.

The presence of background DNA (bgDNA) can hinder the evaluation of DNA evidence at the activity level, especially when the suspect is expected to be retrieved due to their habitual occupation of the investigated environment. Based on real-life casework circumstances, this study investigates the prevalence, composition, origin, and probable transfer routes of bgDNA found on personal items in situations where their owner and person of interest (POI) share the same workspace. Baseline values of bgDNA were evaluated on the participants' personal items. Secondary and higher degree transfer scenarios of non-self DNA deposition were also investigated. The DNA from co-workers and co-inhabiting partners can be recovered from an individual's personal belongings. Non-self DNA present on the hands and deposited on a sterile surface can generate uninformative profiles. The accumulation of foreign DNA on surfaces over time appears to be crucial for the recovery of comparable profiles, resulting in detectable further transfer onto other surfaces. For a thorough evaluation of touch DNA traces at the activity level, it is necessary to collect information not only about DNA transfer probabilities but also about the presence of the POI as part of the 'baseline' bgDNA of the substrates involved.

The presence of background DNA on common entry points to homes

It is becoming increasingly relevant to understand how much DNA is present on, and who might contribute to, samples collected from surfaces frequently targeted for DNA sampling in criminal investigations. This study investigates the presence of such DNA, also termed background DNA (bDNA), on common entry points in five homes occupied by known individuals. Samples were collected from internal and external windowsills and edges within bedrooms and living rooms, as well as front and rear door handles. The findings of this study contribute to our current knowledge on the prevalence of bDNA in the home and will assist scientists in making informed decisions on sampling strategy.

Target-Triggered Polymerization of Branched DNA Enables Enzyme-free and Fast Discrimination of Single-Base Changes.

SummarySingle-base changes lead to important biological and biomedical implications; however, the discrimination of single-base changes from normal DNA always remains a grand challenge. Herein we developed a DNA recognition and amplification system based on artificial branched DNA, namely, target-triggered polymerization (TTP), to realize enzyme-free and fast discrimination of single-base changes. Branched DNA as monomers rapidly polymerized into DNA nanospheres only with the trigger of specific DNA. Our TTP system worked reliably over a wide range of conditions. Remarkably, our TTP system was capable of discriminating base-changing DNA from normal DNA, including distinguishing 1–4 nucleotide changes and positions of single base, which was attributed to the significant amplification of small differences in hybridization thermodynamics and kinetics. We further proposed a theoretical method for calculating the hybridization probability of nucleic acids, which performed highly consistent with experimental results.

Applicability of quantitative polymerase chain reaction (qPCR) assays for common bean authentication in processed food

SummaryFood authentication by quantitative polymerase chain reaction (qPCR) methods assures food quality. The aim was to evaluate three qPCR assays for DNA quantification after heat processing of common bean grains, genus‐specific FAS assay for Phaseolus, species‐specific LEC assay for common bean (Phaseolus vulgaris) and genetically modified (GM) event‐specific FGM assay for Embrapa 5.1 event GM common bean. FAS assay showed high stability among Phaseolus genus samples. Common bean grains were heat‐treated in autoclave (at 120 °C for 15–60 min) and target DNA copy number decreased as processing time increased. Even with DNA degradation, qPCR assays were capable to detect low DNA quantity, and the limit of detection was 100 copy number. Mean efficiency value of FGM assay was 92% in the presence of background DNA. Background DNA did not cause any interference, and 0.39% of GM material can be detected. These qPCR assays are able to quantify common bean in processed food.

Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum.

Fusarium culmorum is a ubiquitous, soil-borne fungus (ascomycete) causing foot and root rot and Fusarium head blight on cereals. It is responsible for yield and quality losses as well as grain contamination with mycotoxins, which are a potential health hazard. An extremely sensitive mitochondrial-based qPCR assay (FcMito qPCR) for quantification of F. culmorum was developed in this study. To provide specificity, the FcMito assay was successfully validated against 85 F. culmorum strains and 53 isolates of 30 other fungal species. The assay efficiency and sensitivity were evaluated against different F. culmorum strains with various amounts of pure fungal DNA and in the presence of background wheat DNA. The results demonstrated the high efficiency of the assay (97.2–106.0%, R2-values > 0.99). It was also shown that, in the presence of background DNA, 0.01 pg of fungal template could be reliably quantified. The FcMito assay was used to quantify F. culmorum DNA using 108 grain samples with different trichothecene levels. A significant positive correlation was found between fungal DNA quantity and the total trichothecene content. The obtained results showed that the sensitivity of the FcMito assay was much higher than the nuclear-based qPCR assay for F. culmorum.

Evaluation of a triplex real-time PCR system to detect the plant-pathogenic molds Alternaria spp., Fusarium spp. and C. purpurea

This article describes the development of a triplex real-time PCR system for the simultaneous detection of three major plant-pathogenic mold genera (Alternaria spp., Fusarium spp. and the species Claviceps purpurea). The designed genus-specific primer–probe systems were validated for sensitivity, specificity and amplification in the presence of background DNA.

An investigation of the presence of DNA on unused laboratory gloves

The presence of background DNA in forensic laboratories is potentially a significant contamination risk to criminal investigations. As part of the laboratory's Environmental Monitoring (EM) program, a preliminary investigation of three brands of laboratory gloves was undertaken to determine the levels of human DNA present on unused gloves from closed and open boxes as well as the origin of stains observed on gloves. The study revealed the presence of DNA on a number of the gloves from closed boxes as well as on some gloves from open boxes. Some observed staining on gloves was indicative of rust. Regular assessment of the presence of human DNA on unused gloves is recommended. In addition, using only certified DNA-free gloves should be considered.

Empirical Evaluation of a New Method for Calculating Signal-to-Noise Ratio for Microarray Data Analysis

Signal-to-noise-ratio (SNR) thresholds for microarray data analysis were experimentally determined with an oligonucleotide array that contained perfect-match (PM) and mismatch (MM) probes based upon four genes from Shewanella oneidensis MR-1. A new SNR calculation, called the signal-to-both-standard-deviations ratio (SSDR), was developed and evaluated, along with other two methods, the signal-to-standard-deviation ratio (SSR) and the signal-to-background ratio (SBR). At a low stringency, the thresholds of the SSR, SBR, and SSDR were 2.5, 1.60, and 0.80 with an oligonucleotide and a PCR amplicon as target templates and 2.0, 1.60, and 0.70 with genomic DNAs as target templates. Slightly higher thresholds were obtained under high-stringency conditions. The thresholds of the SSR and SSDR decreased with an increase in the complexity of targets (e.g., target types) and the presence of background DNA and a decrease in the compositions of targets, while the SBR remained unchanged in all situations. The lowest percentage of false positives and false negatives was observed with the SSDR calculation method, suggesting that it may be a better SNR calculation for more accurate determination of SNR thresholds. Positive spots identified by SNR thresholds were verified by the Student t test, and consistent results were observed. This study provides general guidance for users to select appropriate SNR thresholds for different samples under different hybridization conditions.

Equal Performance of TaqMan, MGB, Molecular Beacon, and SYBR Green-Based Detection Assays in Detection and Quantification of Roundup Ready Soybean

We have tested and compared the performance of 12 different assays representing four different real-time polymerase chain reaction (PCR) chemistries in the context of genetically modified organism detection. Several different molecular beacon, SYBR Green, TaqMan, and MGB assays were designed for the event specific detection and quantification of the 3' integration junction of GTS 40-3-2 (Roundup Ready) soybean. Sensitivity as well as robustness in the presence of background DNA were tested. None of the PCR-based approaches appeared to be significantly better than any of the other, but the molecular beacon assays had the lowest efficiency and also seemed more sensitive to changes in experimental setup.

Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays.

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.