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3.
Nat Commun ; 11(1): 3392, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636376

RESUMO

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Quadruplex G , Estruturas R-Loop , Transcrição Genética , Proteínas Virais/genética , DNA/análise , DNA/química , RNA Polimerases Dirigidas por DNA/química , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , RNA/química , RNA Mensageiro/química , Sítio de Iniciação de Transcrição , Proteínas Virais/química
4.
PLoS One ; 15(7): e0231918, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702012

RESUMO

Determining the concentration of nucleic acids in biological samples precisely and reliably still is a challenge. In particular when only very small sample quantities are available for analysis, the established fluorescence-based methods give insufficient results. Photobleaching is seen as the main reason for this. In this paper we present a method to correct for the photobleaching effect. Using confocal microscopy with single molecule sensitivity, we derived calibration curves from DNA solutions with defined fragment length. We analyzed dilution series over a wide range of concentrations (1 pg/µl-1000 pg/µl) and measured their specific diffusion coefficients employing fluorescence correlation spectroscopy. Using this information, we corrected the measured fluorescence intensity of the calibration solutions for photobleaching effects. We evaluated our method by analyzing a series of DNA mixtures of varying composition. For fragments smaller than 1000 bp, our method allows to determine sample concentrations with high precision in very small sample quantities (< 2 µl with concentrations < 20 pg/µl). Once the technical parameters are determined and remain stable in an established process, our improved calibration method will make measuring molecular biological samples of unknown sequence composition more efficient, accurate and sample-saving than previous methods.


Assuntos
DNA/análise , DNA/química , Microscopia Confocal , Fotodegradação , Difusão , Limite de Detecção , Soluções
5.
PLoS One ; 15(7): e0235054, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609728

RESUMO

Elucidating the diets of insect predators is important in basic and applied ecology, such as for improving the effectiveness of conservation biological control measures to promote natural enemies of crop pests. Here, we investigated the aphid diet of two common aphid predators in Central European agroecosystems, the native Coccinella septempunctata (Linnaeus) and the invasive Harmonia axyridis (Pallas; Coleoptera: Coccinellidae) by means of high throughput sequencing (HTS). For acquiring insights into diets of mobile flying insects at landscape scale minimizing trapping bias is important, which imposes methodological challenges for HTS. We therefore assessed the suitability of three field sampling methods (sticky traps, pan traps and hand-collection) as well as new aphid primers for identifying aphid prey consumption by coccinellids through HTS. The new aphid primers facilitate identification to species level in 75% of the European aphid genera investigated. Aphid primer specificity was high in silico and in vitro but low in environmental samples with the methods used, although this could be improved in future studies. For insect trapping we conclude that sticky traps are a suitable method in terms of minimizing sampling bias, contamination risk and trapping success, but compromise on DNA-recovery rate. The aphid diets of both field-captured ladybird species were dominated by Microlophium carnosum, the common nettle aphid. Another common prey was Sitobion avenae (cereal aphid), which got more often detected in C. septempunctata compared to H. axyridis. Around one third of the recovered aphid taxa were common crop pests. We conclude that sampling methodologies need constant revision but that our improved aphid primers offer currently one of the best solutions for broad screenings of coccinellid predation on aphids.


Assuntos
Afídeos/genética , Besouros/fisiologia , Cadeia Alimentar , Comportamento Predatório , Ração Animal/análise , Animais , Afídeos/classificação , DNA/análise , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Espécies Introduzidas , Especificidade da Espécie
6.
Nat Commun ; 11(1): 3609, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681090

RESUMO

Standard units of measurement are required for the quantitative description of nature; however, few standard units have been established for genomics to date. Here, we have developed a synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA sample, and act as an internal scale that measures quantitative genetics features. Unlike previous spike-ins, the ladder is encoded within a single molecule, and can be equivalently and independently synthesized by different laboratories. We show how the ladder can measure diverse quantitative features, including human genetic variation and microbial abundance, and also estimate uncertainty due to technical variation and improve normalization between libraries. This ladder provides an independent quantitative unit that can be used with any organism, application or technology, thereby providing a common metric by which genomes can be measured.


Assuntos
DNA/análise , DNA/síntese química , Sequência de Bases , DNA/genética , Dosagem de Genes , Biblioteca Gênica , Genômica , Humanos
7.
Forensic Sci Int Genet ; 48: 102346, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32660901

RESUMO

The aggressive nature of the new SARS-2 corona virus now referred to as SARS-CoV-2 ; the seriousness and length of the period of infection; the fast and far-reaching transmissibility via liquid droplets that become air-borne when someone coughs, sneezes or speaks with increasing evidence to support actual airborne transmission; the presence of viral particles especially in body fluids and tissues, of viral positive individuals; and the persistence of the virus on different types of surfaces pose serious concerns for forensic practitioners, including forensic DNA analysts. Many forensic laboratories and law enforcement agencies need to address the inevitable changes that must be made in forensic DNA testing. In this article, we explore the effects of the COVID-19 pandemic on the collection, handling, storage and transport of biological samples for downstream DNA testing. This paper aims to open discussions on the urgency of balancing the need to conduct investigations in order to maintain public order with the requirements of effective biosafety protocols specifically formulated to protect human resources within the forensic science community.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , DNA/análise , Genética Forense , Pandemias , Pneumonia Viral/epidemiologia , Infecções por Coronavirus/virologia , DNA/genética , Bases de Dados Genéticas , Humanos , Pneumonia Viral/virologia
8.
PLoS One ; 15(7): e0235793, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634162

RESUMO

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.


Assuntos
Fracionamento Celular/métodos , Células Endoteliais/química , Vesículas Extracelulares/química , Biomarcadores/análise , Centrifugação/métodos , Precipitação Química , DNA/análise , Células Endoteliais/virologia , Vesículas Extracelulares/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas/análise , RNA/análise
9.
Forensic Sci Int Genet ; 48: 102346, 2020 09.
Artigo em Inglês | MEDLINE | ID: covidwho-642775

RESUMO

The aggressive nature of the new SARS-2 corona virus now referred to as SARS-CoV-2 ; the seriousness and length of the period of infection; the fast and far-reaching transmissibility via liquid droplets that become air-borne when someone coughs, sneezes or speaks with increasing evidence to support actual airborne transmission; the presence of viral particles especially in body fluids and tissues, of viral positive individuals; and the persistence of the virus on different types of surfaces pose serious concerns for forensic practitioners, including forensic DNA analysts. Many forensic laboratories and law enforcement agencies need to address the inevitable changes that must be made in forensic DNA testing. In this article, we explore the effects of the COVID-19 pandemic on the collection, handling, storage and transport of biological samples for downstream DNA testing. This paper aims to open discussions on the urgency of balancing the need to conduct investigations in order to maintain public order with the requirements of effective biosafety protocols specifically formulated to protect human resources within the forensic science community.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , DNA/análise , Genética Forense , Pandemias , Pneumonia Viral/epidemiologia , Infecções por Coronavirus/virologia , DNA/genética , Bases de Dados Genéticas , Humanos , Pneumonia Viral/virologia
10.
PLoS One ; 15(7): e0235500, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614886

RESUMO

INTRODUCTION: Clinical trials often suffer from significant recruitment barriers, poor adherence, and dropouts, which increase costs and negatively affect trial outcomes. The aim of this study was to examine whether making it virtual and reward-based would enable nationwide recruitment, identify patients with variable disease severity, achieve high adherence, and reduce dropouts. METHODS: In a siteless, virtual feasibility study, individuals with atopic dermatitis (AD) were recruited online. During the 8-week study, subjects used their smartphones weekly to photograph target AD lesions, and completed patient-oriented eczema measure (POEM) and treatment use questionnaires. In return, subjects were rewarded every week with personalized lifestyle reports based on their DNA. RESULTS: Over the course of the 11 day recruitment period, 164 (82% women and 18% men) filled in the form to participate, of which 65 fulfilled the inclusion criteria and signed the informed consent. Ten were excluded as they did not complete the mandatory study task of returning the DNA sample. 55 (91% women, 9% men) subjects returned the DNA sample and were enrolled throughout Denmark, the majority outside the Copenhagen capital region in rural areas with relatively low physician coverage. The mean age was 28.5 (SD ±9.5 years, range 18-52 years). The baseline POEM score was 14.5±5.6 (range 6-28). Based on the POEM, 7 individuals had mild, 28 had moderate, 17 had severe, and 3 had very severe eczema. The retention rate was 96% as 53 out of 55 enrolled completed the study. The adherence was very high, and more than 90% of all study tasks were completed. Follow up of 41 subjects showed that 90% would take part again or continue if the study had been longer. CONCLUSION: A virtual trial design enables recruitment with broad geographic reach and throughout the full spectrum of disease severity. Providing personalized genetic reports as a reward seems to contribute to high adherence and retention.


Assuntos
Dermatite Atópica/psicologia , Eczema/patologia , Recompensa , Cooperação e Adesão ao Tratamento , Adolescente , Adulto , DNA/análise , Dermatite Atópica/genética , Dermatite Atópica/patologia , Dermatite Atópica/terapia , Fármacos Dermatológicos/uso terapêutico , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Fototerapia , Índice de Gravidade de Doença , Smartphone , Inquéritos e Questionários , Adulto Jovem
11.
PLoS One ; 15(7): e0235748, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701977

RESUMO

With advances in sequencing technology, a vast amount of genomic sequence information has become available. However, annotating biological functions particularly of non-protein-coding regions in genome sequences without experiments is still a challenging task. Recently deep learning-based methods were shown to have the ability to predict gene regulatory regions from genome sequences, promising to aid the interpretation of genomic sequence data. Here, we report an improvement of the prediction accuracy for gene regulatory regions by using the design of convolution layers that efficiently process genomic sequence information, and developed a software, DeepGMAP, to train and compare different deep learning-based models (https://github.com/koonimaru/DeepGMAP). First, we demonstrate that our convolution layers, termed forward- and reverse-sequence scan (FRSS) layers, integrate both forward and reverse strand information, and enhance the power to predict gene regulatory regions. Second, we assessed previous studies and identified problems associated with data structures that caused overfitting. Finally, we introduce visualization methods to examine what the program learned. Together, our FRSS layers improve the prediction accuracy for gene regulatory regions.


Assuntos
DNA/análise , Genoma , Genômica/métodos , Redes Neurais de Computação , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA/métodos , Software , Animais , DNA/genética , Humanos , Camundongos
12.
Rev. osteoporos. metab. miner. (Internet) ; 12(2): 40-44, abr.-jun. 2020. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-193782

RESUMO

OBJETIVO: Las células madre mesenquimales (MSCs) son atractivas en la terapia regenerativa de patologías humanas. En los modelos murinos, en los que se trasplantan MSCs humanas, es muy importante poder distinguir el origen de las MSCs identificadas en los órganos de ratones. El objetivo de este estudio fue determinar el rendimiento del análisis basado en PCR de secuencias Alu humanas para detectar ADN humano después de la infusión de células madre de médula ósea humana (hBMSCs) en ratones inmunodeficientes. MATERIAL Y MÉTODO: Las hBMSCs se obtuvieron de la cabeza femoral de pacientes sometidos a cirugía de reemplazo de cadera. Se infundieron 106 hBMSCs por vía intravenosa mediante inyección en el seno retro-orbitario de ratones NOD/SCID. Después se evaluó la presencia de ADN humano en pulmón, hígado y hueso. RESULTADOS: En mezclas de ADN in vitro, el ADN humano se detectó fácilmente con una buena relación logarítmica-lineal. De manera similar, cuando se mezclaron osteoblastos humanos y de ratón, se detectaron fácilmente 1-10 células humanas entre 105 células de ratón. Asimismo, se detectó el ADN humano en los pulmones 1 y 7 días después de las infusiones celulares en ratones NOD/SCID. Sin embargo, el ADN humano se detectó de manera inconsistente en el hígado y los huesos. CONCLUSIÓN: La detección de secuencias Alu es un procedimiento eficaz para detectar ADN humano. Los resultados confirman que la mayoría de las hBMSCs inyectadas por vía intravenosa quedan atrapadas en los pulmones. Por lo tanto, de cara al tratamiento de trastornos esqueléticos, se necesitan procedimientos para aumentar la migración de dichas células al hueso


OBJETIVE: Mesenchymal stem cells (MSCs) are commonly used in regenerative therapy of human diseases. In murine models, in which human MSCs are transplanted, distinguishing the origin of the identified MSCs in the organs of mice is important. The objective of this study was to determine the performance of PCR-based analysis of human Alu sequences to detect human DNA after infusion of human bone marrow stem cells (hBMSCs) in immunodeficient mice. MATERIAL AND METHOD: HBMSCs were obtained from the femoral head of patients undergoing hip replacement surgery. 106 hBMSCs were infused intravenously by injection into the retro-orbital sinus of NOD/SCID mice. The presence of human DNA in lung, liver and bone was then assessed. RESULTS: In in vitro DNA mixtures, human DNA was easily detected with a good logarithmic-linear relationship. Similarly, when human and mouse osteoblasts were mixed, 1-10 cells were easily detected among 105 mouse cells. Likewise, human DNA was detected in the lungs 1 and 7 days after cell infusions in NOD/SCID mice. However, human DNA was inconsistently detected in the liver and bones. CONCLUSION: Detecting Alu sequences is an effective procedure to observe human DNA. The results confirm that most intravenously injected hBMSCs are trapped in the lungs. Thus, for the treatment of skeletal disorders, procedures are needed to increase the migration of these cells to the bone


Assuntos
Humanos , Camundongos , Movimento Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , DNA/análise , Reação em Cadeia da Polimerase , Modelos Animais
13.
PLoS One ; 15(6): e0235216, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32598374

RESUMO

A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA's performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.


Assuntos
Arabidopsis/genética , DNA/análise , Fusarium/genética , Doenças das Plantas/genética , Arabidopsis/microbiologia , DNA/genética , Fusarium/patogenicidade , Técnicas de Amplificação de Ácido Nucleico , Doenças das Plantas/microbiologia , Temperatura
14.
PLoS One ; 15(6): e0234745, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32544213

RESUMO

PCR inhibitors are a formidable problem to the study of aged, degraded, and/or low copy number DNA. As a result, there is a need to find alternate methods that ameliorate the efficacy of PCR. In this study, we attempted to use genetic methods to identify the species of salmonid (Oncorhynchus spp.) remains recovered from archaeological sites along the Feather River located in northern California, United States. In the process of doing so, we compared the efficacy of a PCR enhancer cocktail called "PEC-P" and a reagent rich PCR recipe called "rescue PCR" over standard PCR. Across all treatments (full concentration and 1:10 dilute eluates subjected to standard PCR, PEC-P, and rescue PCR) species identification was possible for 74 of 93 archaeological fish specimens (79.6%). Overall, six of the 93 samples (6.5%) consistently yielded species identification across all treatments. The species of ten specimens (10.8%) were uniquely identified from amplicons produced with either PEC-P or rescue PCR or both. Notably, the species of seven samples (7.5%) were uniquely identified with standard PCR over the alternative treatments. Considering both full concentration and 1:10 dilute eluates (N = 186), standard PCR performed as well as PEC-P (p = 0.1451) and rescue (p = 0.6753). Yet, considering results from full concentration eluates alone (N = 93), PEC-P (60.2%) outperformed both standard PCR (44.1%; p = 0.0277) and rescue PCR (40.9%; p = 0.0046). Stochasticity observed in our study cautions us against choosing a "best" performing method of those explored here and suggests their respective potentials to improve success may be sample dependent. When working with samples compromised by PCR inhibitors, it is useful to have alternative methodologies for subduing the problem. Both PEC-P and rescue PCR represent useful alternative methods for the study of aged, degraded, and/or low copy number DNA samples compromised by PCR inhibitors.


Assuntos
DNA/análise , Oncorhynchus/genética , Reação em Cadeia da Polimerase/métodos , Animais , Arqueologia , Osso e Ossos/metabolismo , DNA/metabolismo , DNA Mitocondrial/análise , DNA Mitocondrial/metabolismo , Fatores de Tempo
15.
PLoS Comput Biol ; 16(6): e1007770, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516306

RESUMO

A longstanding goal of regulatory genetics is to understand how variants in genome sequences lead to changes in gene expression. Here we present a method named Bayesian Annotation Guided eQTL Analysis (BAGEA), a variational Bayes framework to model cis-eQTLs using directed and undirected genomic annotations. We used BAGEA to integrate directed genomic annotations with eQTL summary statistics from tissues of various origins. This analysis revealed epigenetic marks that are relevant for gene expression in different tissues and cell types. We estimated the predictive power of the models that were fitted based on directed genomic annotations. This analysis showed that, depending on the underlying eQTL data used, the directed genomic annotations could predict up to 1.5% of the variance observed in the expression of genes with top nominal eQTL association p-values < 10-7. For genes with estimated effect sizes in the top 25% quantile, up to 5% of the expression variance could be predicted. Based on our results, we recommend the use of BAGEA for the analysis of cis-eQTL data to reveal annotations relevant to expression biology.


Assuntos
Biologia Computacional/métodos , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Algoritmos , Teorema de Bayes , Mapeamento Cromossômico , DNA/análise , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Humano , Genômica , Genótipo , Humanos , Anotação de Sequência Molecular , Monócitos/metabolismo , Software
16.
Gene ; 754: 144860, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32531457

RESUMO

Arunachal Pradesh, the largest state of North-East India covers almost 60.93% of the Eastern Himalayan hotspot. Fish diversity and species identification is utmost important for fisheries management. But, in some cases morphological characteristics based identification is difficult for a non-specialist to perform. In view of the above, the present study emphasized on the assessment of DNA barcoding, phylogenetics and genetic diversity of fish species in the Ranganadi River, Arunachal Pradesh, India. India. Arunachal Pradesh, the largest state of North-East India covers almost 60.93% of the Eastern Himalayan hotspot. Altogether 114 specimens, representing 22 species, belonging to 3 orders and 5 families were successfully barcoded and found to be 98-100% identical from both GenBank and BOLD databases. Out of these 22 fish species, it was found that one species assessed was Endangered, three species as Near Threatened and one species as Vulnerable. A Neighbour Joining (NJ) tree was constructed using Rstudio for the purpose of a phylogenetic analysis of the identified species. The barcoding gap analysis using K2P, P-distance and Jukes-Cantor was done to detect the presence of cryptic species and barcoding success. The nucleotide base composition and genetic distance analysis were also performed, using MEGA 6.0. DNA Sequence Polymorphism v6.12.03 analysis revealed the nucleotide diversity (p) and haplotype diversity (Hd). The Hd for the whole dataset was found to be 0.975, which showed high genetic diversity in the Ranganadi River. Both morphological key identifying characters and molecular data corroborated the phylogenetic analysis. This COI barcode library, generated in the present study, not only helped in species identification and molecular study, but also in cryptic species identification.


Assuntos
Biodiversidade , Código de Barras de DNA Taxonômico , DNA/análise , Peixes/classificação , Peixes/genética , Variação Genética , Filogenia , Animais , DNA/genética , Água Doce , Rios
17.
Food Chem ; 331: 127163, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32593037

RESUMO

Herein, a surface plasmon resonance (SPR) enhanced DNA biosensor has been developed for real-time detection of donkey meat marker using biotinylated reporter and streptavidin functionalized gold nanostars (Stre@GNSs). Compared to the direct detection assay, this sandwich format for the enhancement of the signal, resulted in 6-folds orders increase in the sensitivity. Target DNA could be detected with the lower limit of quantification (LLOQ) of 1.0 nM with a relative standard deviation (RSD, n = 3) of 0.85%. In addition, the fabricated SPR sensor showed good selectivity for the target analyte over full complementary, single-base mismatch, three base-mismatch and non-complementary oligonucleotides. Finally, the proposed sensor was successfully applied for detection of donkey meat adulteration with various percentages in homemade beef sausage, as a real sample. The results indicated that the proposed biosensor provides a high specificity, easy, good sensitivity and fast approach for identification of donkey meat adulteration in food samples.


Assuntos
Técnicas Biossensoriais/métodos , Equidae/genética , Contaminação de Alimentos/análise , Carne/análise , Animais , Técnicas Biossensoriais/instrumentação , DNA/análise , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Ouro/química , Limite de Detecção , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Espectrofotometria Ultravioleta , Estreptavidina/química , Ressonância de Plasmônio de Superfície/métodos
18.
Proc Natl Acad Sci U S A ; 117(24): 13421-13427, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482858

RESUMO

Although the backlog of untested sexual assault kits in the United States is starting to be addressed, many municipalities are opting for selective testing of samples within a kit, where only the most probative samples are tested. We use data from the San Francisco Police Department Criminalistics Laboratory, which tests all samples but also collects information on the samples flagged by sexual assault forensic examiners as most probative, to build a standard machine learning model that predicts (based on covariates gleaned from sexual assault kit questionnaires) which samples are most probative. This model is embedded within an optimization framework that selects which samples to test from each kit to maximize the Combined DNA Index System (CODIS) yield (i.e., the number of kits that generate at least one DNA profile for the criminal DNA database) subject to a budget constraint. Our analysis predicts that, relative to a policy that tests only the samples deemed probative by the sexual assault forensic examiners, the proposed policy increases the CODIS yield by 45.4% without increasing the cost. Full testing of all samples has a slightly lower cost-effectiveness than the selective policy based on forensic examiners, but more than doubles the yield. In over half of the sexual assaults, a sample was not collected during the forensic medical exam from the body location deemed most probative by the machine learning model. Our results suggest that electronic forensic records coupled with machine learning and optimization models could enhance the effectiveness of criminal investigations of sexual assaults.


Assuntos
Vítimas de Crime , Ciências Forenses/economia , Aplicação da Lei/métodos , Delitos Sexuais , Manejo de Espécimes/economia , Adulto , Análise Custo-Benefício , Vítimas de Crime/estatística & dados numéricos , DNA/análise , Bases de Dados de Ácidos Nucleicos , Feminino , Ciências Forenses/estatística & dados numéricos , Humanos , Aprendizado de Máquina , Masculino , São Francisco , Delitos Sexuais/estatística & dados numéricos , Manejo de Espécimes/estatística & dados numéricos
19.
J Vis Exp ; (160)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32568232

RESUMO

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.


Assuntos
Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , DNA/análise , Fezes/química , Adulto , Análise de Dados , Fluorescência , Humanos , Pessoa de Meia-Idade , Sangue Oculto , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Fatores de Risco , Temperatura
20.
Leg Med (Tokyo) ; 46: 101713, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32442862

RESUMO

An evaluation of a Rapid DNA system was performed using buccal swab samples and mock Disaster Victim Identification (DVI) samples collected postmortem. The allelic ladder success rate was 90% and samples analyzed simultaneously with this allelic ladder were used for further analysis. Sample success rate of the Rapid DNA system for buccal swab samples, and blood and muscle DVI samples were calculated. Success rates of buccal swab samples were 100% and 75% using cassettes preloaded with all reagents suitable for high- and low-DNA content samples, respectively. Success rates of fresh DVI samples were 80% to 100%. Success rates of putrefied DVI samples varied widely between 0% and 20% and 50% to 80% depending on cassette and sample types. Conventional DNA analysis was performed for comparison with the results of the Rapid DNA system. DNA quantity and degradation of human DNA were measured using quantitative polymerase chain reaction. DVI samples that yielded more than 1 ng/µL of DNA when extracted with conventional protocols were suitable for analysis using cassettes for both high- and low-DNA content samples. DVI samples with less than 0.1 ng/µL of DNA were suitable only for analysis using cassettes for low-DNA content samples. All alleles called and exported by the Expert system software implemented in the Rapid DNA system were concordant with allele calls made by conventional capillary electrophoresis DNA analysis.


Assuntos
Impressões Digitais de DNA/métodos , Vítimas de Desastres , Medicina Legal/métodos , Mucosa Bucal , DNA/análise , Humanos , Manejo de Espécimes
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