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1.
Chem Commun (Camb) ; 55(65): 9709-9712, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31353371

RESUMO

We functionalize nucleic acid nanostructures with click chemistry (for attachment of cargos) and a photocleavable linker (for release). We demonstrate cargo attachment using a fluorescein dye and release using UV trigger from an RNA three-way junction, a DNA star motif and a DNA tetrahedron. Such multifunctional nucleic acid nanostructures have potential in targeted drug delivery.


Assuntos
DNA/química , Portadores de Fármacos/química , Fluoresceínas/química , Corantes Fluorescentes/química , Nanoestruturas/química , RNA/química , Animais , Fagos Bacilares/genética , Sequência de Bases , Bovinos , Química Click , DNA/sangue , DNA/síntese química , DNA/efeitos da radiação , Portadores de Fármacos/síntese química , Portadores de Fármacos/efeitos da radiação , Fluorescência , Nanoestruturas/efeitos da radiação , Conformação de Ácido Nucleico , RNA/sangue , RNA/síntese química , RNA/efeitos da radiação , Raios Ultravioleta
2.
Nat Commun ; 10(1): 2548, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186427

RESUMO

Epigenetic processes, including DNA methylation (DNAm), are among the mechanisms allowing integration of genetic and environmental factors to shape cellular function. While many studies have investigated either environmental or genetic contributions to DNAm, few have assessed their integrated effects. Here we examine the relative contributions of prenatal environmental factors and genotype on DNA methylation in neonatal blood at variably methylated regions (VMRs) in 4 independent cohorts (overall n = 2365). We use Akaike's information criterion to test which factors best explain variability of methylation in the cohort-specific VMRs: several prenatal environmental factors (E), genotypes in cis (G), or their additive (G + E) or interaction (GxE) effects. Genetic and environmental factors in combination best explain DNAm at the majority of VMRs. The CpGs best explained by either G, G + E or GxE are functionally distinct. The enrichment of genetic variants from GxE models in GWAS for complex disorders supports their importance for disease risk.


Assuntos
Metilação de DNA/genética , DNA/sangue , Interação Gene-Ambiente , Estudos de Coortes , Epigênese Genética , Feminino , Sangue Fetal , Genótipo , Humanos , Recém-Nascido , Masculino , Gravidez , Fatores de Risco
3.
Cardiovasc Diabetol ; 18(1): 49, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992036

RESUMO

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with a hypercoagulable state and increased neutrophil extracellular traps formation (NETosis). We investigated predictors of NETosis and cell death markers in circulating blood and their association with a prothrombotic state in T2DM. METHODS: In a cross-sectional study involving 113 T2DM patients aged 63.7 ± 8.2 years, we investigated citrullinated histone H3 (H3Cit), cell-free deoxyribonucleic acid (cfDNA), myeloperoxidase, neutrophil elastase, and inflammation markers, along with thrombin generation (TG), plasma clot lysis time (CLT), clot permeability (Ks) and fibrinolysis inhibitors. RESULTS: On multivariate logistic regression analysis adjusted for age and gender, predictors of high H3Cit (≥ 7.36 ng/mL, upper quartile) were: glycated hemoglobin (HbA1c) ≥ 7.0% and interleukin-6. Interleukin-6 was also found to be a predictor of high cfDNA (≥ 2.84 µg/mL, upper quartile) along with glucose. Citrullinated histone H3 and cfDNA correlated positively with CLT and inversely with Ks, while TG associated solely with cfDNA. These associations were not seen with myeloperoxidase and neutrophil elastase. Patients with previous myocardial infarction (n = 21, 18.6%) had higher H3Cit (+108%, p < 0.001) and cfDNA (+45%, p = 0.022). On multivariable analysis adjusted for potential confounders, H3Cit and cfDNA, along with plasminogen activator inhibitor-1 and concomitant cardiovascular disease, were predictors of CLT. Citrullinated histone H3 alone was a predictor of Ks and only cfDNA was a predictor of peak thrombin generated. CONCLUSIONS: In T2DM, NETosis detectable in circulating blood is associated with inflammatory state and a prothrombotic state, especially hypofibrinolysis.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Armadilhas Extracelulares/metabolismo , Fibrinólise , Trombose/sangue , Idoso , Biomarcadores/sangue , Glicemia/metabolismo , Ácidos Nucleicos Livres/sangue , Citrulinação , Estudos Transversais , DNA/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Hemoglobina A Glicada/metabolismo , Histonas/sangue , Humanos , Mediadores da Inflamação/sangue , Elastase de Leucócito/sangue , Masculino , Pessoa de Meia-Idade , Peroxidase/sangue , Fatores de Risco , Trombina/metabolismo , Trombose/diagnóstico , Trombose/etiologia
4.
Analyst ; 144(9): 3088-3093, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30919845

RESUMO

In this work, a label-free fluorescence biosensor was proposed for simple detection of the Kras wild type by using the three way DNA junction-driven catalyzed hairpin assembly strategy. In this system, a three-way DNA junction probe (JP) and two hairpin probes (H1 and H2) were designed. In the presence of the Kras wild type, an autocatalytic DNA machine can be activated. This leads to the generation of numerous free G-rich sequences, which can associate with a fluorescent dye N-methylmesoporphyrin IX (NMM) to yield an amplified fluorescence signal for the target detection. This sensing platform showed a high sensitivity towards the Kras wild type with a detection limit as low as 2.7 fM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits an excellent selectivity for the Kras wild type compared with other interference DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the Kras wild type in a real biological sample with satisfactory results, suggesting that this method is promising for simple and early clinical diagnosis of genetic diseases. Thanks to its simplicity, cost-effectiveness, and ultrasensitivity, our proposed sensing strategy provides a universal platform for the detection of other genetic diseases by substituting the target-recognition element.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , DNA/sangue , Proteínas Proto-Oncogênicas p21(ras)/genética , Espectrometria de Fluorescência/métodos , Sequência de Bases , DNA/química , DNA/genética , Sondas de DNA/genética , Fluorescência , Corantes Fluorescentes/química , Quadruplex G , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Mesoporfirinas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico
5.
Analyst ; 144(8): 2649-2655, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30843550

RESUMO

In this work, we propose an ultrasensitive fluorescence strategy for DNA detection. This method utilizes a molecular beacon (MB), a hairpin probe (HP), and an enzyme to trigger dual-cycling reactions (cycles I and II). In cycle I, the target is repeatedly used to amplify the fluorescence emission through hybridizations with the MB and cleavage reactions achieved by the enzyme. In cycle II, hybridization reactions between the HP and a segment of the MB continuously regenerate the target to trigger more cycle I reactions, leading to an enhanced fluorescent signal. The detection limit of the method is determined to be as low as 50 fM within 45 min, which is 2 to 3 orders of magnitude lower than that of the conventional fluorescence strategies. The method also shows a high selectivity over mismatched and random DNA sequences. The signal amplification mechanism of the strategy offers insights into constructing efficient and ultrasensitive biosensors for various applications.


Assuntos
DNA/sangue , Genes p53 , Espectrometria de Fluorescência/métodos , Animais , Técnicas Biossensoriais/métodos , Bovinos , DNA/química , DNA/genética , Desoxirribonuclease I/química , Fluorescência , Corantes Fluorescentes/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico
6.
Methods Mol Biol ; 1972: 213-219, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30847794

RESUMO

Quantitative analysis of DNA methylation patterns is of special importance in several developmental and pathological situations. The development of simple and robust methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly reproducible CE-UV method for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolyzed samples were dried and successively dissolved with water and then injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4:1, v/v) allowed a baseline analytes separation within 12 min. Precision tests indicated an elevated reproducibility with an inter-assay CV of 1.98%.


Assuntos
Metilação de DNA/genética , Eletroforese Capilar/métodos , Genoma Humano , Raios Ultravioleta , Calibragem , DNA/sangue , Humanos , Padrões de Referência
7.
Anal Bioanal Chem ; 411(10): 2101-2109, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790017

RESUMO

Presently, most reported electrochemical biosensors, for highly sensitive and selective detection of nucleic acid, still require multiple, time-consuming assembly steps and high-consumption DNA probes as well as lack good performance in human serum, which greatly limit their applicability. Herein, an easy-to-fabricate electrochemical DNA biosensor constructed by assembly of bovine serum albumin (BSA) followed with direct incubation of amplified products has been proposed. This method combined terminal deoxynucleoside transferase (TdTase)-mediated isothermal amplification and polyHRP catalysis to achieve dual-signal enhancement, and was featured with low-density DNA monolayer for its employment of only 2 nM capture probes. Surprisingly, based on the low-density DNA monolayer, the steric hindrance effect of polyHRP could effectively restrain the background compared with HRP, which further pushes the signal-to-noise (S/N) ratio to 70 than that of most currently available methods. Additionally, this strategy also showed favorable specificity and powerful anti-interference in human serum, and thus potentially attractive for diagnosis of diseases.


Assuntos
Técnicas Biossensoriais/métodos , DNA/sangue , Técnicas Eletroquímicas/métodos , Animais , Sequência de Bases , Técnicas Biossensoriais/instrumentação , Bovinos , DNA/análise , DNA Nucleotidilexotransferase/química , Sondas de DNA/química , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Humanos , Limite de Detecção , Soroalbumina Bovina/química
8.
Mol Diagn Ther ; 23(2): 291-299, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30712216

RESUMO

Non-invasive prenatal diagnosis (NIPD) is based on fetal DNA analysis starting from a simple peripheral blood sample, thus avoiding risks associated with conventional invasive techniques. During pregnancy, the fetal DNA increases to approximately 3-13% of the total circulating free DNA in maternal plasma. The very low amount of circulating cell-free fetal DNA (ccffDNA) in maternal plasma is a crucial issue, and requires specific and optimized techniques for ccffDNA purification from maternal plasma. In addition, highly sensitive detection approaches are required. In recent years, advanced ccffDNA investigation approaches have allowed the application of non-invasive prenatal testing (NIPT) to determine fetal sex, fetal rhesus D (RhD) genotyping, aneuploidies, micro-deletions and the detection of paternally inherited monogenic disorders. Finally, complex and innovative technologies such as digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) (exhibiting higher sensitivity and/or the capability to read the entire fetal genome from maternal plasma DNA) are expected to allow the detection, in the near future, of maternally inherited mutations that cause genetic diseases. The aim of this review is to introduce the principal ccffDNA characteristics and their applications as the basis of current and novel NIPT.


Assuntos
DNA/sangue , Diagnóstico Pré-Natal/métodos , Ácidos Nucleicos Livres/sangue , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Testes Genéticos , Humanos
9.
Nanoscale ; 11(8): 3633-3638, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30741288

RESUMO

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 µL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.


Assuntos
DNA Bacteriano/análise , Endonucleases Flap/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Chlamydia trachomatis/genética , DNA/análise , DNA/sangue , DNA/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/sangue , DNA de Cadeia Simples/metabolismo , Humanos , Limite de Detecção , Ácidos Nucleicos/sangue , Ácidos Nucleicos/metabolismo , Compostos Orgânicos/química
10.
Proc Natl Acad Sci U S A ; 116(2): 641-649, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30593563

RESUMO

Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3 -/- mice carrying Dnase1l3 +/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.


Assuntos
Ácidos Nucleicos Livres/sangue , Fragmentação do DNA , DNA/sangue , Endodesoxirribonucleases/metabolismo , Motivos de Nucleotídeos , Animais , Ácidos Nucleicos Livres/genética , DNA/genética , Endodesoxirribonucleases/genética , Feminino , Feto/metabolismo , Deleção de Genes , Camundongos , Camundongos Knockout , Gravidez
11.
Diabetes Obes Metab ; 21(1): 95-102, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30073765

RESUMO

AIM: To evaluate whether ß cells continue to undergo death in the later stages of type 1 diabetes (T1D). MATERIALS AND METHODS: Fasting banked sera from a cross-section of 90 participants in the T1D Exchange Registry with longstanding T1D (median duration of 9 years) were analysed. Subjects were determined to be C-peptide (-) or (+) based on mixed-meal tolerance testing. Results were compared with 54 adult non-diabetic controls. Stimulated samples were assayed in a subset of subjects. Levels of unmethylated and methylated preproinsulin (INS) DNA were analysed using digital droplet PCR. RESULTS: Fasting and stimulated circulating unmethylated INS DNA levels were increased among both C-peptide (-) and C-peptide (+) subjects with longstanding T1D compared with non-diabetic controls (P < 0.01). Consistent with prior reports, unmethylated INS DNA values correlated with methylated INS DNA values, which were also elevated among T1D subjects (P < 0.001). There was wide variation in the effects of mixed-meal stimulation on DNA levels, with fasting values in the highest quartiles decreasing with stimulation (P < 0.05). CONCLUSIONS: These results could reflect ongoing ß cell death in individuals with longstanding T1D, even in the absence of detectable C-peptide production, suggesting that therapies targeting ß cell survival could be beneficial among individuals with longstanding T1D.


Assuntos
DNA , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/epidemiologia , Insulina , Precursores de Proteínas , Adulto , Peptídeo C/sangue , Estudos de Casos e Controles , DNA/sangue , DNA/genética , Metilação de DNA , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Humanos , Insulina/sangue , Insulina/genética , Células Secretoras de Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Precursores de Proteínas/genética , Adulto Jovem
12.
Int J Mol Med ; 43(1): 155-166, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30365064

RESUMO

Congenital hypopituitarism (CH) is a relatively rare disease that is characterized by the deficiency of one or more hormones secreted by the pituitary gland, which leads to metabolic disorders, amenorrhea and infertility. However, the underlying molecular mechanisms of CH have not yet been fully elucidated. The present study evaluated the genome­wide methylation level of whole blood DNA in 12 patients with CH and 12 age­matched controls using Illumina Human Methylation 450 array, in order to determine the roles of epigenetic regulation in the pathogenesis of CH. The results demonstrated that the methylation levels of 51 CpG sites were significantly different between the patients with CH and the controls. Functional enrichment analysis identified that the aberrant methylated genes were enriched in gene sets associated with metabolic or cellular process, immune system process and reproduction. In addition, two CpG sites on genes LIM domain kinase 2 (LIMK2) and piwi­like RNA­mediated gene silencing 2 (PIWIL2), which are involved in spermatogenesis and/or testicular development, were identified to be hypermethylated in male patients with CH. The hypermethylation of these sites was further validated in another 40 patients with CH and 40 matched controls with a quantitative bisulfite pyrosequencing method, and the methylation levels of these two loci demonstrated promising diagnostic capacities for CH. The present results suggested that aberrant methylation of genes may be involved in the pathogenesis of CH, and hypermethylation of LIMK2 and PIWIL2 may contribute to the infertility of male patients with CH. Further studies are required to elucidate the underlying mechanisms of the epigenetic regulation of these genes.


Assuntos
Células Sanguíneas/metabolismo , Metilação de DNA/genética , DNA/sangue , Estudo de Associação Genômica Ampla , Hipopituitarismo/sangue , Hipopituitarismo/genética , Adulto , Estudos de Casos e Controles , Análise por Conglomerados , Ilhas de CpG/genética , DNA/genética , Genoma Humano , Humanos , Hipopituitarismo/diagnóstico , Masculino , Curva ROC , Adulto Jovem
13.
PLoS One ; 13(12): e0208915, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571772

RESUMO

Epigenome-wide association studies seek to identify DNA methylation sites associated with clinical outcomes. Difference in observed methylation between specific cell-subtypes is often of interest; however, available samples often comprise a mixture of cells. To date, cell-subtype estimates have been obtained from mixed-cell DNA data using linear regression models, but the accuracy of such estimates has not been critically assessed. We evaluated linear regression performance for cell-subtype specific methylation estimation using a 450K methylation array dataset of both mixed-cell and cell-subtype sorted samples from six healthy males. CpGs associated with each cell-subtype were first identified using t-tests between groups of cell-subtype sorted samples. Subsequent reduced panels of reliably accurate CpGs were identified from mixed-cell samples using an accuracy heuristic (D). Performance was assessed by comparing cell-subtype specific estimates from mixed-cells with corresponding cell-sorted mean using the mean absolute error (MAE) and the Coefficient of Determination (R2). At the cell-subtype level, methylation levels at 3272 CpGs could be estimated to within a MAE of 5% of the expected value. The cell-subtypes with the highest accuracy were CD56+ NK (R2 = 0.56) and CD8+T (R2 = 0.48), where 23% of sites were accurately estimated. Hierarchical clustering and pathways enrichment analysis confirmed the biological relevance of the panels. Our results suggest that linear regression for cell-subtype specific methylation estimation is accurate only for some cell-subtypes at a small fraction of cell-associated sites but may be applicable to EWASs of disease traits with a blood-based pathology. Although sample size was a limitation in this study, we suggest that alternative statistical methods will provide the greatest performance improvements.


Assuntos
Linhagem da Célula/genética , Metilação de DNA/genética , DNA/sangue , Epigenômica , Células Sanguíneas , Ilhas de CpG/genética , DNA/genética , Epigênese Genética , Feminino , Humanos , Modelos Lineares , Masculino , Transdução de Sinais/genética , Resultado do Tratamento
14.
Anal Chim Acta ; 1043: 81-88, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30392672

RESUMO

We demonstrate for the first time the development of a universal DNAzyme-based bioluminescent sensor for label-free detection of various biomolecules including DNAzyme and DNA. The presence of DNAzyme may induce the cyclic cleavage of riboadenosine (rA)-containing substrates, and the subsequent digestion of the cleaved substrates by exonuclease III (Exo III) releases abundant AMPs to initiate cyclic AMP pyrophosphorylation-ATP depyrophosphorylation for the generation of an enhanced bioluminescence signal. This sensor can real-time monitor the DNAzyme activity with a detection limit of 3.16 × 10-12 M. Moreover, the DNAzyme may be divided into two subunits for sensitive detection of target DNA. In the presence of target DNA, the two separated subunits may assemble into an active DNAzyme which can catalyze the cyclic cleavage of substrates and initiate the digestion of cleaved substrates by Exo III for the generation of an enhanced bioluminescence signal. This sensor can sensitively detect target DNA with a detection limit of 3.31 × 10-12 M. Importantly, this bioluminescent sensor can achieve a zero-background signal, and its output signal originates from the release of AMP for the generation of self-illuminating light emission without the requirement of either the external labels or the reporting reagents.


Assuntos
DNA Catalítico/metabolismo , DNA/análise , Medições Luminescentes/métodos , Monofosfato de Adenosina/metabolismo , Animais , Biocatálise , Bovinos , DNA/sangue , DNA/metabolismo , Eletroforese em Gel de Ágar , Exodesoxirribonucleases/metabolismo , Limite de Detecção , Especificidade por Substrato
16.
Int J Mol Sci ; 19(8)2018 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-30081520

RESUMO

Due to the addressability and programmability, DNA has been applied not merely in constructing static elegant nanostructures such as two dimensional and three dimensional DNA nanostructures but also in designing dynamic nanodevices. Moreover, DNA could combine with hydrophobic organic molecules to be a new amphiphilic building block and then self-assemble into nanomaterials. Of particular note, a recent state-of-the-art research has turned our attention to the amphiphilic DNA organic hybrids including small molecule modified DNA (lipid-DNA, fluorescent molecule-DNA, etc.), DNA block copolymers, and DNA-dendron hybrids. This review focuses mainly on the development of their self-assembly behavior and their potential application in nanomaterial and biomedicine. The potential challenges regarding of the amphiphilic DNA organic hybrids are also briefly discussed, aiming to advance their practical applications in nanoscience and biomedicine.


Assuntos
DNA/sangue , Polímeros/química , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hibridização de Ácido Nucleico
17.
Clin Biochem ; 60: 71-76, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30092181

RESUMO

OBJECTIVES: Thirty-six blood group systems are listed by the International Society of Blood Transfusion, containing almost 350 antigens. Most of these result from a single nucleotide polymorphism (SNP). Serology is the standard method for blood group typing. However, this technique has some limitations and cannot respond to the growing demand of blood product typing for a large number of antigens. Here we describe a blood group genotyping assay directly from whole blood samples using Next-Generation Sequencing (NGS), allowing the simultaneous identification of 15 SNPs associated with the blood group systems of 95 patients in a single run. DESIGN AND METHOD: After an automated DNA extraction, targets are amplified by multiplex polymerase chain reaction (PCRm). Two panels addressing 9 groups have been developed (MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, and Colton), one for 8 SNPs, the other for 7 SNPs. For each sample, both panels corresponding to 14 amplicons (1 amplicon containing 2 SNPs) are pooled. Then a dual-indexed library is generated from each pool by linking Illumina adaptors directly onto amplicons, followed by sequencing using the MiSeq platform (Illumina). RESULTS: In a single experiment, 95 blood donor samples have been sequenced for the genes of interest. Among the 1425 targeted single nucleotide polymorphisms, 1420 were identified by sequencing, reflecting a coverage of 99.65%. The obtained data shows a good correlation (99% for all SNPs) with other blood group typing methods. Depending on the allele pairs analyzed, correlations vary between 97.12 and 100%. CONCLUSION: Next-Generation sequencing would supplement serological and molecular techniques and, in the near future, could replace it with complete and fast results acquisition for pre-screening and identification of rare blood bags.


Assuntos
Antígenos de Grupos Sanguíneos/genética , DNA/sangue , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Alelos , Humanos , Reação em Cadeia da Polimerase Multiplex , Polimorfismo de Nucleotídeo Único
18.
Carbohydr Polym ; 197: 100-108, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30007594

RESUMO

We examined the properties of the nanocomposite γ-Fe2O3@Chi@Pani as an adsorbent of deoxyribonucleic acid (DNA). As a model system, we used an aqueous solution of salmon sperm DNA, whose decreasing concentration was followed by monitoring the 260 nm UV-vis absorption. After adjusting the data collected to a Langmuir isotherm curve, we estimated the adsorption capacity (qe) of the nanocomposite as 49.5 mg/g. We also observed that the kinetic model of the DNA capture presents a mixed character, with both chemical mechanisms and intraparticle diffusion processes involved. When the MNC was used to extract the DNA from complex samples (human blood), a capture rate of 80 ng/µL was achieved, with the collected fraction exhibiting good quality, as evaluated by PCR analysis and electrophoresis assays. These results suggest that the γ-Fe2O3@Chi@Pani nanocomposite is a promising adsorbent for use in protocols for purification of DNA from complex samples.


Assuntos
Compostos de Anilina/química , Quitosana/química , DNA/isolamento & purificação , Compostos Férricos/química , Nanopartículas de Magnetita/química , Nanocompostos/química , DNA/sangue , DNA/química , Humanos
19.
Talanta ; 188: 685-690, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029432

RESUMO

Taking advantage of the homogeneous and heterogeneous electrochemical biosensors, a simple, sensitive, and selective electrochemical biosensor is constructed by combining entropy-driven amplification (EDA) with DNA walker. This electrochemical biosensor realizes the biorecognition and EDA operation in homogeneous solution, which is beneficial to improve the recognition and amplification efficiency. A two-leg DNA walker generated by EDA can walk on the surface of gold electrode for cleaving the immobilized substrate DNA and releasing the electroactive labels, giving rise to a significant decrease of the electrochemical signal. The immobilization of the electroactive labels ensures the reproducibility and reliability of the biosensor. The present cascade amplification assay can be applied to detect target DNA with a detection limit of 0.29 fM, and base mutations can be easily distinguished. Moreover, the proposed electrochemical biosensor shows a satisfactory performance for the detection of target DNA in human serum. Thus, the novel electrochemical biosensor holds promising potential for a future application in disease diagnosis.


Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/química , DNA/sangue , Técnicas Eletroquímicas/métodos , Ácidos Nucleicos Imobilizados/sangue , Nanoestruturas/química , DNA/metabolismo , Eletrodos , Ouro/química , Humanos , Chumbo/química , Limite de Detecção , Azul de Metileno/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes
20.
Mol Biol Rep ; 45(5): 925-929, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29982892

RESUMO

Analysis of DNA polymorphisms are the primary technique used for personal identification in forensic cases. However, DNA samples collected as evidence from crime scenes are usually degraded by environmental, physical, and chemical factors, which may interfere with the PCR analysis and, consequently, personal identification. Whole genome amplification (WGA) is a useful method to amplify genomic DNA from samples containing low quantity and poor quality of DNA, and it approach that shows promise to overcome the limited small fragments based upon random fragmentation by universal priming sites. In this study, we describe the use of WGA to genotype 15 short tandem repeat (STR) loci from dried blood samples irradiated with different types of ultraviolet (UV) light (UVA, UVB, and UVC). The result showed that UVC was the most damaging to DNA, followed by UVB and UVA. Samples exposed to UVA could be genotyped for all STR loci with or without WGA. For UVB and UVC irradiated blood samples, a greater number of STR loci could be genotyped after WGA. Although it hard to amplified a few higher molecular weight alleles, overall, the WGA method was useful in genotyping template DNA of poor quality but low quantity.


Assuntos
DNA/efeitos da radiação , Técnicas de Genotipagem/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Manchas de Sangue , DNA/sangue , DNA/genética , Teste em Amostras de Sangue Seco , Genética Forense , Genoma Humano , Humanos , Repetições de Microssatélites
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