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1.
J Forensic Leg Med ; 67: 24-27, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377682

RESUMO

BACKGROUND: A mass grave is any site that containing two or more associated corpses, at random or on purpose placed, of people who have died as a result of extra-judicial or random executions, not including those people who have died from armed confrontations or known major catastrophes. CASE PRESENTATION: The purpose of this paper is to explain how to reconstruct a biological profile of decomposed or skeletonized bodies and clarify the efforts done by the Libyan scientist after 2011 revolution and to set a reference for other researcher. The alleged location of the grave, as well as the alleged number and identities of the persons buried in the grave were obtained exclusively from witnesses' and relatives' testimonies. CONCLUSION: As the testimonies said, the grave was located at the alleged location and seven skeletons were exhumed. Also, the osteological and DNA study made investigators to identify the exhumed skeletons. And the dental analysis support the identification of a seven man alleged to have been buried in the grave, 7 victims were discovered.


Assuntos
Osso e Ossos/patologia , Impressões Digitais de DNA , Exumação , Adulto , Determinação da Idade pelo Esqueleto , Sepultamento , DNA/isolamento & purificação , Antropologia Forense , Odontologia Legal , Humanos , Líbia , Masculino , Repetições de Microssatélites , Determinação do Sexo pelo Esqueleto , Dente/química , Guerra
2.
Anthropol Anz ; 76(4): 333-351, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31322643

RESUMO

Skeletal remains are among the most difficult types of samples encountered in forensic DNA casework and historical investigations due to prolonged exposure to environmental insults. DNA extracted from bone often is degraded, in low quantities, and contains co-purified inhibitors from the surrounding soil and/or burial vault material. When sexually dimorphic skeletal elements are not recovered, determining the sex of a decedent can be challenging. With unidentified human skeletal remains, genetic data often are evaluated in concert with anthropological analyses, as well as other types of metadata, to improve confidence in making associations or for positive identifications. This study evaluated a multi-faceted molecular genetic approach to increasing the amount of data that can be recovered from degraded skeletal remains. Results demonstrate that using a newer-generation multiplex (GlobalFiler™) with an expanded set of highly discriminatory DNA markers - combined with co-amplification of three different sex-determining loci, one additional PCR cycle, and testing multiple cuttings from the same bone or multiple regions within a skeleton - can improve reliability and accuracy in skeletal remains identifications by providing data concordance.


Assuntos
Restos Mortais , Impressões Digitais de DNA , DNA , Antropologia , Consenso , DNA/isolamento & purificação , Humanos , Repetições de Microssatélites , Reprodutibilidade dos Testes
3.
Sud Med Ekspert ; 62(2): 22-25, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31213587

RESUMO

The objective of the present study was to optimize genotyping of nuclear DNA contained in human hair. The most efficient procedures for DNA isolation and typing are described taking into consideration the hair growth phase, the epithelial tissue conditions, and the number of nuclei in the near-root ends of the hair. The recommendations for the expert interpretation of the results of the molecular-genetic investigations are proposed.


Assuntos
Núcleo Celular/genética , Impressões Digitais de DNA , DNA/isolamento & purificação , Genótipo , Cabelo/citologia , Humanos
4.
J Forensic Leg Med ; 66: 107-112, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31254968

RESUMO

More than 200 men were killed at the Koricani Cliffs on Mount Vlasic in central Bosnia during the Bosnian war. The location of this mass grave remained unknown for a long time following the war, until 2008, when the Missing Persons Institute discovered a site containing the remains of approximately 60 individuals. Later, in September 2017, a new mass grave was identified at this location that had not been robbed and skeletal remains remained close to the location where the victims had died. This grave was also unique, by definition, as it was a primary inhumation site, but with a high degree of commingling and disarticulation, typical of secondary inhumation locations. The exhumation team found the first remains in this grave approximately one and a half meters beneath the rocks, while the extent of the commingling resulted in necessary modifications to standardized exhumation protocols. The search and recovery process primarily focused on skulls, groups of bones that remained in clothing, and any bones that were still connected to each other. In total, 86 skulls, 137 groups of bones that had at least 2 bones connected, and a couple of hundred small bones that could not be appointed to individuals, were retrieved. The material was taken to the Sejkovaca Identification Centre where the team took over 1,300 DNA samples for analysis and are now awaiting the results.


Assuntos
Osso e Ossos/patologia , Exumação , Guerra , Osso e Ossos/química , Bósnia e Herzegóvina , Sepultamento , DNA/isolamento & purificação , Antropologia Forense , Humanos , Masculino , Solo
5.
J Chem Ecol ; 45(5-6): 429-439, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31152352

RESUMO

Animal classification is primarily based on morphological characters, even though these may not be the first to diverge during speciation. In many cases, closely related taxa are actually difficult to distinguish based on morphological characters alone, especially when there is no substantial niche separation. As a consequence, the diversity of certain groups is likely to be underestimated. Lepidoptera -moths and butterflies- represent the largest group of herbivorous insects. The extensive diversification in the group is generally assumed to have its origin in the spectacular radiation of flowering plants and the resulting abundance of ecological niches. However, speciation can also occur without strong ecological divergence. For example, reproductive isolation can evolve as the result of divergence in mate preference and the associated pheromone communication system. We combined pheromone trapping and genetic analysis to elucidate the evolutionary relationships within a complex of primitive moth species (Lepidoptera: Eriocraniidae). Mitochondrial and nuclear DNA markers provided evidence that Eriocrania semipurpurella, as currently defined by morphological characters, includes three cryptic species in Northern and Western Europe. Male moths of these cryptic species, as well as of the closely related E. sangii, exhibited relative specificity in terms of their attraction to specific ratios of two major pheromone components, (2S,6Z)-nonen-2-ol and (2R,6Z)-nonen-2-ol. Our data suggest strong assortative mating in these species in the absence of apparent niche separation, indicating that Eriocrania moths may represent an example of non-ecological speciation. Finally, our study argues in favour of combining pheromone investigations and DNA barcoding as powerful tools for identifying and delimitating species boundaries.


Assuntos
Mariposas/genética , Atrativos Sexuais/química , Animais , DNA/isolamento & purificação , DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Variação Genética , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/classificação , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/genética , Masculino , Mitocôndrias/genética , Filogenia , Atrativos Sexuais/metabolismo
6.
Ann Lab Med ; 39(5): 478-487, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31037867

RESUMO

BACKGROUND: Plasma epidermal growth factor receptor (EGFR) mutation tests are less invasive than tissue EGFR mutation tests. We determined which of two kits is more efficient: cobas EGFR Mutation test v2 (cobasv2; Roche Molecular Systems, Pleasanton, CA, USA) or PANAMutyper-R-EGFR (Mutyper; Panagene, Daejeon, Korea). We also evaluated whether pleural effusion supernatant (PE-SUP) samples are assayable, similar to plasma samples, using these two kits. METHODS: We analyzed 156 plasma and PE-SUP samples (31 paired samples) from 116 individuals. We compared the kits in terms of accuracy, assessed genotype concordance (weighted κ with 95% confidence intervals), and calculated Spearman's rho between semi-quantitatively measured EGFR-mutant levels (SQIs) measured by each kit. We also compared sensitivity using 47 EGFR-mutant harboring samples divided into more-dilute and less-dilute samples (dilution ratio: ≥ or <1:1,000). RESULTS: cobasv2 tended to have higher accuracy than Mutyper (73% vs 69%, P=0.53), and PE-SUP samples had significantly higher accuracy than plasma samples (97% vs 55-71%) for both kits. Genotype concordance was 98% (κ=0.92, 0.88-0.96). SQIs showed strong positive correlations (P<0.0001). In less-dilute samples, accuracy and sensitivity did not differ significantly between kits. In more-dilute samples, cobasv2 tended to have higher sensitivity than Mutyper (43% vs 20%, P=0.07). CONCLUSIONS: The kits have similar performance in terms of EGFR mutation detection and semi-quantification in plasma and PE-SUP samples. cobasv2 tends to outperform Mutyper in detecting less-abundant EGFR-mutants. PE-SUP samples are assayable using either kit.


Assuntos
Receptores ErbB/genética , Derrame Pleural/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA/isolamento & purificação , DNA/metabolismo , Receptores ErbB/sangue , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Derrame Pleural/sangue , Derrame Pleural/genética , Kit de Reagentes para Diagnóstico , Adulto Jovem
7.
Sensors (Basel) ; 19(9)2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060322

RESUMO

A new voltammetric DNA sensor has been developed for doxorubicin determination on the platform of a glassy carbon electrode (GCE) covered with electropolymerized Azure B film and physically adsorbed native DNA. The redox properties of polymeric Azure B were monitored at various pH and scan rates. DNA application decreased the peak currents related to polymeric and monomeric forms of the dye, whereas incubation in doxorubicin solution partially restored the peaks in accordance with the drug and DNA concentration. The relative shift of the cathodic peak current caused by doxorubicin depended on the nominal DNA concentration and its application mode. In optimal conditions, the DNA sensor makes it possible to determine between 0.1 µM to 0.1 nM doxorubicin (limit of detection 7×10-11 M). The DNA sensor was tested on commercial doxorubicin formulations and on artificial samples the mimicked electrolyte content of human serum.


Assuntos
Técnicas Biossensoriais , DNA/isolamento & purificação , Doxorrubicina/isolamento & purificação , Nanotubos de Carbono/química , Corantes Azur/química , DNA/química , Doxorrubicina/química , Técnicas Eletroquímicas/métodos , Humanos , Oxirredução , Polímeros/química
8.
Forensic Sci Int ; 300: 99-105, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31085432

RESUMO

A novel method for detection and visualization of latent DNA using Diamond™ Nucleic Acid Dye (DD) staining has been developed. Applying DD to an object has the real potential to visualize DNA on a substrate from which a DNA profile can be generated, but it is important to determine whether this staining will adversely affect other forensic investigational techniques and vice versa. The aim of this study was to examine the interactions between staining a fingermark to detect DNA and then generate a DNA profile in combination with several standard latent fingermark enhancement methods. Six common fingerprint enhancements processes were chosen; (1) black powder, (2) black magnetic powder, (3) red magnetic powder, (4) white powder, (5) aluminum powder and (6) cyanoacrylate fuming. For all six methods, mark enhancement was carried out before DD staining and vice versa. DD is effective in detection of DNA in the presence of both aluminum and white finger mark powders and DD does not compromise the subsequent detection of ridge patterns if DD is applied first. Whilst magnetic powders could be used to successfully enhance latent fingermarks even after DD had been applied to them, latent DNA could not be observed in the marks irrespective of whether magnetic powder was applied before or after DD treatment. Magnetic powders did not adversely affect the profiling of DNA present in the marks. The application of DD to fingermarks did not adversely affect the enhancement of fingermarks using cyanoacrylate fuming. Whilst fluorescent particles resembling cells stained with DD were observed in marks either post-treated or pre-treated with cyanoacrylate vapor, DNA amplification and profiling was not successful. While it may be important in particular investigations to collect DNA profiles from latent fingermarks with continuous ridges and clear minutiae, the main utility of the technique described here would be in relation to investigations where enhancement has resulted in only partial or smudged marks. The results presented here indicate that if it is desirable to visualize latent DNA on an object but it is also planned to treat the object with cyanoacrylate vapor or magnetic powders then it is important to apply DD first, record the location of DNA and then apply the mark enhancement technique. For aluminum and white powder mark treatments such precautions are not important.


Assuntos
Corantes , DNA/isolamento & purificação , Dermatoglifia , Cianoacrilatos , Impressões Digitais de DNA , Feminino , Humanos , Masculino , Repetições de Microssatélites , Microscopia , Reação em Cadeia da Polimerase , Pós , Tato , Volatilização
9.
J Coll Physicians Surg Pak ; 29(5): 483-485, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31036126

RESUMO

The aim of this study was to explore the feasibility of non-invasive prenatal genetic diagnosis of ß-thalassemia with small fragments of cell-free fetal DNA (cffDNA) in peripheral blood of pregnant women. It was an observational study carried out at Department of Obstetrics, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, China, from January 2016 to March 2018. A total of 40 pregnant women, who were likely to give birth to babies with severe ß-thalassemia, were selected, and ß-globin genotype of the fetus was non-invasively detected by cffDNA in peripheral blood of their mothers. Small fragments of cffDNA from all specimens were successfully amplified. Compared with the results of traumatic prenatal diagnosis, 37 cases (92.50%) were diagnosed and 3 cases (7.50%) were misdiagnosed. The cffDNA in maternal plasma can be used for non-invasive prenatal genetic diagnosis of ß-thalassemia, and is worthy of promotion.


Assuntos
Ácidos Nucleicos Livres/sangue , DNA/sangue , DNA/genética , Amplificação de Genes/genética , Gravidez/sangue , Diagnóstico Pré-Natal/métodos , Globinas beta/genética , Talassemia beta/diagnóstico , Adulto , Ácidos Nucleicos Livres/genética , DNA/isolamento & purificação , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/genética , Feto/irrigação sanguínea , Testes Genéticos , Humanos , Gestantes , Talassemia beta/genética
10.
Appl Microbiol Biotechnol ; 103(11): 4575-4584, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31001745

RESUMO

The identification of animal species in feed and feedstuffs is important for detecting contamination and fraudulent replacement of animal components that might cause health and economic problems. A novel multiplex assay, based on xMAP technology and the generic detection of closely related species, was developed for the simultaneous differential detection of avian, fish, and ruminant DNA in products. Universal primers and probes specific to avian, fish, or ruminant species were designed to target a conserved mitochondrial DNA sequence in the 12S ribosomal RNA gene (rRNA). The assay specificity was validated using samples of 27 target and 10 nontarget animal species. The limits of detection of the purified DNA were determined to be 0.2 pg/µL-0.1 ng/µL by testing the meat samples of six species and four feedstuffs. The detection sensitivity of the experimental mixtures was demonstrated to be 0.01% (weight percentage). The assay's suitability for practical application was evaluated by testing feed samples; unlabeled animal ingredients were detected in 32% of the 56 samples. The assay differentially detected the three targeted categories of animal species in less than 2 h, reflecting improvements in speed and efficiency. Based on these results, this novel multiplex xMAP assay provides a reliable and highly efficient technology for the routine detection of animal species in feed and other products for which this information is needed.


Assuntos
Ração Animal/análise , DNA/isolamento & purificação , Contaminação de Alimentos , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Aves , DNA/genética , Primers do DNA/genética , Peixes , Sondas de Oligonucleotídeos/genética , RNA Ribossômico/genética , Ruminantes , Sensibilidade e Especificidade
11.
Forensic Sci Int ; 299: 161-167, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31022675

RESUMO

EZ1 Advanced XL (Qiagen) is an instrument designed to purify automatically nucleic acids from a wide variety of samples: different protocols for DNA extraction from forensic samples are pre-loaded into special EZ1 Advanced XL DNA Investigator Cards. The present study focuses on DNA extraction from biological samples (blood, saliva, sperm) mixed with 3 different types of soils (loam, silt, sandy), using a modified version of the Manufacturer standard protocol. In order to create several traces in the soil, we used biological samples (blood, saliva, sperm) from known donors. Quantification data, reliability and trends in STRs typing success rates using two different commercial multiplexes were evaluated. EZ1 modified DNA extraction protocol allows to recover DNA free of inhibitors and in good quantity for downstream applications.


Assuntos
DNA/isolamento & purificação , Genética Forense/instrumentação , Solo/química , Análise Química do Sangue , Impressões Digitais de DNA , Genética Forense/métodos , Humanos , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Saliva/química , Sêmen/química , Manejo de Espécimes
12.
Biosens Bioelectron ; 133: 160-168, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30933710

RESUMO

An efficient and new electrochemical biosensor for detection of DNA damage, induced by the interaction of the hybrid anti-cancer compound (7ESTAC01) with DNA, was studied by differential pulse voltammetry (DPV). The biosensor consists of a Stem-Loop DNA (SL-DNA) probe covalently attached to the gold electrode (GE) surface that hybridizes to a complementary DNA strand (cDNA) to form a double-stranded DNA (dsDNA). The interaction and DNA damage induced by 7ESTAC01 was electrochemically studied based on the oxidation signals of the electroactive nucleic acids on the surface of the GE by DPV. As a result, the SL-DNA/GE and dsDNA/GE were tested with the reduced 7ESTAC01, showing the voltammetric signal of guanine and adenine, increase in the presence of 7ESTAC01. Under optimum conditions, the dsDNA/GE biosensor exhibited excellent DPV response in the presence of 7ESTAC01. The bonding interaction between 7ESTAC01 and calf thymus DNA (ctDNA) was confirmed by UV-Vis absorption spectroscopy, dynamic simulations (performed to investigate the DNA structure under physiological conditions), and molecular docking. Theoretical results showed the presence of hydrogen bonding and intercalation in the minor groove of DNA, involving hydrophobic interactions.


Assuntos
Antineoplásicos/química , Técnicas Biossensoriais , DNA/isolamento & purificação , Técnicas Eletroquímicas , Antineoplásicos/farmacologia , DNA/química , DNA/genética , Dano ao DNA/efeitos dos fármacos , DNA Complementar/química , DNA Complementar/genética , Ouro/química , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Sequências Repetidas Invertidas/genética , Simulação de Acoplamento Molecular , Oxirredução/efeitos dos fármacos , Raios Ultravioleta
13.
Biosens Bioelectron ; 133: 243-249, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30981134

RESUMO

Here, we propose a new two-layer three-dimensional (3-D) DNA walker sensor with highly integrated entropy-driven and enzyme-powered reactions for the first time. The 3-D DNA walker sensor is constructed by assembling densely carboxyfluorescein-labeled single strand oligonucleotides (inner-layer tracks) and nucleic acid complex S (outer-layer tracks) on a microparticle. In the presence of the target, outer and inner tracks are activated in turn, thereby releasing a great deal of the signal reporters for signal reading. As a result, our 3-D DNA walker sensor can realize the target detection in the range from 2 pM to 5 nM within one hour. Besides, the specific walker sensor can clearly distinguish even one-base mismatched target analogue. More importantly, our walker sensor can also test the target in human serum samples in the concentrations as low as 0.1 nM, which provides a bridge between real sample detection and clinical application. Certainly, this smart strategy could also be generalized to any target of interest by proper design.


Assuntos
Técnicas Biossensoriais , DNA/isolamento & purificação , Técnicas Eletroquímicas , HIV/isolamento & purificação , DNA/química , DNA/genética , Entropia , Ouro/química , HIV/patogenicidade , Humanos , Oligonucleotídeos/química
14.
Sensors (Basel) ; 19(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974797

RESUMO

Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopic technique in which the Raman scattering signal strength of molecules, absorbed by rough metals or the surface of nanoparticles, experiences an exponential growth (10³-106 times and even 1014-1015 times) because of electromagnetic or chemical enhancements. Nowadays, SERS has attracted tremendous attention in the field of analytical chemistry due to its specific advantages, including high selectivity, rich informative spectral properties, nondestructive testing, and the prominent multiplexing capabilities of Raman spectroscopy. In this review, we present the applications of state-of-the-art SERS for the detection of DNA, proteins and drugs. Moreover, we focus on highlighting the merits and mechanisms of achieving enhanced SERS signals for food safety and clinical treatment. The machine learning techniques, combined with SERS detection, are also indicated herein. This review concludes with recommendations for future studies on the development of SERS.


Assuntos
Técnicas Biossensoriais , DNA/isolamento & purificação , Proteínas/isolamento & purificação , Análise Espectral Raman , DNA/química , Humanos , Nanopartículas/química , Proteínas/química , Detecção do Abuso de Substâncias/métodos , Propriedades de Superfície
15.
Lab Chip ; 19(9): 1657-1664, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30931470

RESUMO

Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (µCAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng µl-1. Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng µl-1, which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.


Assuntos
Centrifugação/instrumentação , Precipitação Química , DNA/análise , DNA/isolamento & purificação , Dispositivos Lab-On-A-Chip , DNA/genética , Genoma Viral/genética , HIV-1/genética , Técnicas de Amplificação de Ácido Nucleico
16.
Nat Biotechnol ; 37(6): 651-656, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011178

RESUMO

Nanopore DNA sequencing is limited by low base-calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage from 100 to 200 mV, with a frequency of 200 Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.7 ± 0.5% with a constant driving voltage improves to 79.3 ± 0.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.


Assuntos
DNA Helicases/genética , DNA/genética , Nanoporos , Porinas/genética , Algoritmos , DNA/isolamento & purificação , DNA Helicases/química , Mycobacterium smegmatis/genética , Porinas/química , Análise de Sequência de DNA/métodos
17.
Nano Lett ; 19(4): 2668-2673, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30896178

RESUMO

Fluorescence microscopy enables simultaneous observation of the dynamics of single molecules in a large region of interest. Most traditional techniques employ either the geometry or the color of single molecules to uniquely identify (or barcode) different species of interest. However, these techniques require complex sample preparation and multicolor hardware setup. In this work, we introduce a time-based amplification-free single-molecule barcoding technique using easy-to-design nucleic acid strands. A dye-labeled complementary reporter strand transiently binds to the programmed nucleic acid strands to emit temporal intensity signals. We program the DNA strands to emit uniquely identifiable temporal signals for molecular-scale fingerprinting. Since the reporters bind transiently to DNA devices, our method offers relative immunity to photobleaching. We use a single universal reporter strand for all DNA devices making our design extremely cost-effective. We show DNA strands can be programmed for generating a multitude of uniquely identifiable molecular barcodes. Our technique can be easily incorporated with the existing orthogonal methods that use wavelength or geometry to generate a large pool of distinguishable molecular barcodes thereby enhancing the overall multiplexing capabilities of single-molecule imaging.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA/isolamento & purificação , DNA/química , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Fotodegradação
18.
Mol Ecol Resour ; 19(4): 863-876, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30901128

RESUMO

DNA metabarcoding can contribute to improving cost-effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time-consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column-based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic-based enzymatic protocol (BEAD), and a 313-bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7-14 than 1-7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA/genética , Monitorização de Parâmetros Ecológicos/métodos , Água Doce/química , Metagenômica/métodos , Animais , DNA/isolamento & purificação , Invertebrados/classificação , Invertebrados/genética
19.
BMC Genomics ; 20(1): 216, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871467

RESUMO

BACKGROUND: Target enrichment is a critical component of targeted deep next-generation sequencing for the cost-effective and sensitive detection of mutations, which is predominantly performed by either hybrid selection or PCR. Despite the advantages of efficient enrichment, PCR-based methods preclude the identification of PCR duplicates and their subsequent removal. Recently, this limitation was overcome by assigning a unique molecular identifier(UMI) to each template molecule. Currently, several commercial library construction kits based on PCR enrichment are available for UMIs, but there have been no systematic studies to compare their performances. In this study, we evaluated and compared the performances of five commercial library kits from four vendors: the Archer® Reveal ctDNA™ 28 Kit, NEBNext Direct® Cancer HotSpot Panel, Nugen Ovation® Custom Target Enrichment System, Qiagen Human Comprehensive Cancer Panel(HCCP), and Qiagen Human Actionable Solid Tumor Panel(HASTP). RESULTS: We evaluated and compared the performances of the five kits using 50 ng of genomic DNA for the library construction in terms of the library complexity, coverage uniformity, and errors in the UMIs. While the duplicate rates for all kits were dramatically decreased by identifying unique molecules with UMIs, the Qiagen HASTP achieved the highest library complexity based on the depth of unique coverage indicating superb library construction efficiency. Regarding the coverage uniformity, the kits from Nugen and NEB performed the best followed by the kits from Qiagen. We also analyzed the UMIs, including errors, which allowed us to adjust the depth of unique coverage and the length required for sufficient complexity. Based on these comparisons, we selected the Qiagen HASTP for further performance evaluations. The targeted deep sequencing method based on PCR target enrichment combined with UMI tagging sensitively detected mutations present at a frequency as low as 1% using 6.25 ng of human genomic DNA as the starting material. CONCLUSION: This study is the first systematic evaluation of commercial library construction kits for PCR-based targeted deep sequencing utilizing UMIs. Because the kits displayed significant variability in different quality metrics, our study offers a practical guideline for researchers to choose appropriate options for PCR-based targeted sequencing and useful benchmark data for evaluating new kits.


Assuntos
Biomarcadores/análise , DNA/análise , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico/normas , DNA/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Reação em Cadeia da Polimerase/normas
20.
Methods Mol Biol ; 1963: 15-19, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875039

RESUMO

DNA isolated from ancient bones and teeth comprises a mixture of microbial contamination and DNA from the organism under study. In addition, analyses of ancient human remains are often complicated by contamination with present-day human DNA, which can be introduced during excavation and subsequent handling of the specimens. In most cases, the relative abundance of contaminant DNA is much greater than that of the target organism. Here we present two techniques for reducing the proportion of contaminant DNA in bones and teeth. The first and most efficient technique uses a sodium hypochlorite (bleach) pretreatment to destroy contaminant DNA that may be bound or otherwise attached to the surface of bone/tooth powder. The second, less destructive pretreatment uses a phosphate buffer to release surface-bound DNA.


Assuntos
Osso e Ossos/metabolismo , Contaminação por DNA , DNA/análise , DNA/isolamento & purificação , Fosfatos/química , Hipoclorito de Sódio/química , Dente/metabolismo , Animais , DNA/química , Descontaminação , Homem de Neandertal , Reação em Cadeia da Polimerase , Manejo de Espécimes/métodos
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