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1.
Invest Ophthalmol Vis Sci ; 60(14): 4904-4914, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31770435

RESUMO

Purpose: Uveal melanoma is a common primary intraocular malignancy accompanied by high mortality. Previous evidence has highlighted the implication of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in uveal melanoma. Accordingly, we further uncovered the possible role of lncRNA plasmacytoma variant translocation 1 gene (PVT1) and microRNA-17-3p (miR-17-3p) in uveal melanoma. Methods: A series of experiments were performed to examine the relationship among lncRNA PVT1, miR-17-3p, and murine double minute clone 2 oncoprotein (MDM2). Afterward, gain- and loss-of-function approaches were used with uveal melanoma cells to verify the role of lncRNA PVT1, miR-17-3p, and MDM2 in the tumorigenesis and development of uveal melanoma. Results: Highly expressed lncRNA PVT1 and MDM2, yet lowly expressed miR-17-3p, were identified in ocular uveal melanoma tissues versus normal adjacent tissues. Then, dual luciferase reporter gene assay, RNA binding protein immunoprecipitation, and RNA pull-down assays showed that lncRNA PVT1 specifically bound to miR-17-3p, and that MDM2 was a target gene of miR-17-3p. Gain- and loss-of-function studies elucidated that silencing of lncRNA PVT1 or overexpression of miR-17-3p resulted in decreased MDM2 expression and increased transcriptional activity of p53, in addition to inhibiting uveal melanoma cell proliferation, migration, and invasion, yet promoted cell apoptosis in vitro. In addition, lncRNA PVT1 silencing or miR-17-3p overexpression was noted to inhibit tumor growth in vivo. Conclusions: Downregulation of lncRNA PVT1 could potentially promote miR-17-3p expression to suppress tumorigenesis and development of uveal melanoma by activating the p53 signaling pathway through binding to MDM2.


Assuntos
Inativação Gênica/fisiologia , Melanoma/prevenção & controle , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Longo não Codificante/genética , Neoplasias Uveais/prevenção & controle , Animais , Western Blotting , Movimento Celular/fisiologia , Sobrevivência Celular , Primers do DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Genes Reporter , Hibridização in Situ Fluorescente , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Ativação Transcricional , Transplante Heterólogo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Neoplasias Uveais/metabolismo
2.
Parasitol Res ; 118(12): 3469-3478, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31691003

RESUMO

Blastocystis is a prevalent parasite that has a wide distribution. In order to design HRM real-time PCR, primers were selected from SSU rRNA gene to amplify specific fragment with different melting temperatures for each subtype of Blastocystis. Subsequently, HRM real-time PCR was performed and melting curve analysis was done by Rotor-Gene Q software. The results of HRM real-time PCR was then compared with sequence results of "barcoding region" of SSU rRNA gene of Blastocystis. To evaluate sensitivity of test, 10-fold serial dilutions of the parasite were prepared from ~ 106 to 1 parasite per mL of stool sample and were investigated by HRM real-time PCR. In order to determine specificity of method, HRM real-time PCR was done for some microorganisms and Blastocystis-negative stool samples. In silico analysis showed that all seventeen subtypes of Blastocystis were distinguish. In vitro analysis revealed that the test discriminated subtypes with specific melting temperatures.


Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/diagnóstico , Primers do DNA/química , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Testes Diagnósticos de Rotina , Fezes/parasitologia , Humanos , Sensibilidade e Especificidade
3.
Molecules ; 24(19)2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591283

RESUMO

Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.


Assuntos
Aptâmeros de Nucleotídeos/química , Primers do DNA/química , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/síntese química , Primers do DNA/síntese química , DNA de Cadeia Simples/isolamento & purificação , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/química , Reação em Cadeia da Polimerase
4.
J Dairy Sci ; 102(11): 9702-9710, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31477297

RESUMO

Monitoring Staphylococcus aureus with high sensitivity is very important for ensuring milk quality and food safety. In this study, we used a rapid nucleic acid isothermal amplification method, saltatory rolling circle amplification (SRCA), for the detection of Staph. aureus in milk. The results of the SRCA method can be assessed visually by the presence of white precipitate or by fluorescence measurement. Thirteen Staph. aureus strains and 31 non-Staph. aureus strains were used to evaluate the specificity of SRCA. The method exhibited excellent detection of Staph. aureus genomic DNA at a concentration of 7.8 × 101 fg/µL when assessed by visible precipitate, and at 7.8 × 100 fg/µL when detected by fluorescence after addition of the fluorochrome SYBR Green I. In artificially inoculated milk, the detection limits of SRCA were 5.6 × 102 cfu/mL by precipitate and 5.6 × 101 cfu/mL by fluorescence, respectively. Compared with conventional PCR approaches, the SRCA assay achieved at least 100-fold higher sensitivity. Moreover, the sensitivity, specificity, and accuracy of the SRCA-based system were calculated to be 100.00, 97.73, and 97.78%, respectively. These results indicate that SRCA has potential application as a sensitive and visual technique for the detection of Staph. aureus in milk.


Assuntos
Mastite Bovina/diagnóstico , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/isolamento & purificação , Animais , Bovinos , DNA/metabolismo , Primers do DNA/química , DNA Complementar/química , DNA de Cadeia Simples/metabolismo , Limite de Detecção , Mastite Bovina/microbiologia , Técnicas de Amplificação de Ácido Nucleico/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética
5.
Extremophiles ; 23(6): 747-757, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31489482

RESUMO

16S rRNA gene profiling is a powerful method for characterizing microbial communities; however, no universal primer pair can target all bacteria and archaea, resulting in different primer pairs which may impact the diversity profile obtained. Here, we evaluated three pairs of high-throughput sequencing primers for characterizing archaeal communities from deep-sea sediments and permafrost soils. The results show that primer pair Arch519/Arch915 (V4-V5 regions) produced the highest alpha diversity estimates, followed by Arch349f/Arch806r (V3-V4 regions) and A751f/AU1204r (V5-V7 regions) in both sample types. The archaeal taxonomic compositions and the relative abundance estimates of archaeal communities are influenced by the primer pairs. Beta diversity of the archaeal community detected by the three primer pairs reveals that primer pairs Arch349f/Arch806r and Arch519f/Arch915r are biased toward detection of Halobacteriales, Methanobacteriales and MBG-E/Hydrothermarchaeota, whereas the primer pairs Arch519f/Arch915r and A751f/UA1204r are biased to detect MBG-B/Lokiarchaeota, and the primers pairs Arch349f/Arch806r and A751f/UA1204r are biased to detect Methanomicrobiales and Methanosarcinales. The data suggest that the alpha and beta diversities of archaeal communities as well as the community compositions are influenced by the primer pair choice. This finding provides researchers with valuable experimental insight for selection of appropriate archaeal primer pairs to characterize archaeal communities.


Assuntos
Archaea , Primers do DNA , DNA Arqueal , Sedimentos Geológicos/microbiologia , Pergelissolo/microbiologia , Microbiologia do Solo , Archaea/classificação , Archaea/genética , Primers do DNA/química , Primers do DNA/genética , DNA Arqueal/química , DNA Arqueal/genética
6.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
7.
Anal Chim Acta ; 1081: 193-199, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446958

RESUMO

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5'-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme.


Assuntos
Primers do DNA/genética , DNA Viral/análise , DNA Polimerase Dirigida por DNA/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Primers do DNA/química , DNA Viral/genética , Desoxirribonuclease HindIII/química , Geobacillus stearothermophilus/enzimologia , Vírus da Hepatite B/genética , Sequências Repetidas Invertidas , Hibridização de Ácido Nucleico , Estudo de Prova de Conceito , Sensibilidade e Especificidade
8.
PLoS One ; 14(7): e0219978, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329612

RESUMO

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteína 1 de Ligação a X-Box/genética , Animais , Bovinos , Células Cultivadas , Primers do DNA/química , Estresse do Retículo Endoplasmático , Feminino , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real/normas , Suínos , Porco Miniatura , Proteína 1 de Ligação a X-Box/química , Proteína 1 de Ligação a X-Box/metabolismo
9.
BMC Res Notes ; 12(1): 371, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262345

RESUMO

OBJECTIVE: The resurgence of lumpy skin disease virus isolates of different genotypic natures abolishes the accuracy of assays that target either vaccine or field strain genome. The aim of the present study was to develop a universal real-time PCR assay using TaqMan chemistry to cover field, vaccine, and recombinant strains of lumpy skin disease virus isolates. RESULTS: The PCR assay was designed based on a LSDV044 target region that offers a unique identification locus to facilitate the sensitive and specific detection of all isolates known to date. The efficiency of amplification, determined over five orders of magnitude, was 93%, with the standard deviation remaining in the range of 0.11-0.23. Evaluation of the assay repeatability on three different days revealed that the inter-run variability ranged from 0.83 to 1.22 over five repetitions across three runs. This new screening assay is proposed as a fast, efficient, and sensitive tool that can be employed in the basic or applied surveillance studies regardless of the genotype. Moreover, the assay can be used for the routine laboratory testing of animal samples during eradication programs for lumpy skin disease.


Assuntos
Primers do DNA/química , DNA Viral/genética , Vírus da Doença Nodular Cutânea/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Vacinas Virais/química , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , Genótipo , Doença Nodular Cutânea/diagnóstico , Doença Nodular Cutânea/imunologia , Doença Nodular Cutânea/prevenção & controle , Doença Nodular Cutânea/virologia , Vírus da Doença Nodular Cutânea/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vacinas Atenuadas , Vacinas Virais/imunologia
10.
PLoS One ; 14(7): e0220081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339936

RESUMO

Knowledge on the trophic interactions among predators and their prey is important in order to understand ecology and behaviour of animals. Traditionally studies on the diet composition of insectivorous bats have been based on the morphological identification of prey remains, but the accuracy of the results has been hampered due to methodological limitations. Lately, the DNA metabarcoding and High Throughput Sequencing (HTS) techniques have changed the scene since they allows prey identification to the species level, ultimately giving more precision to the results. Nevertheless, the use of one single primer set to amplify faecal DNA produces biases in the assessed dietary composition. Three horseshoe bats overlap extensively in their distribution range in Europe: Rhinolophus euryale, R. hipposideros and R. ferrumequinum. In order to achieve the deepest insight on their prey list we combined two different primers. Results showed that the used primers were complementary at the order and species levels, only 22 out of 135 prey species being amplified by both. The most frequent prey of R. hipposideros belonged to Diptera and Lepidoptera, to Lepidoptera in R. euryale, and Lepidoptera, Diptera and Coleoptera in R. ferrumequinum. The three bats show significant resource partitioning, since their trophic niche overlap is not higher than 34%. Our results confirm the importance of combining complementary primers to describe the diet of generalist insectivorous bats with amplicon metabarcoding techniques. Overall, each primer set showed a subset of the prey composition, with a small portion of the total prey being identified by both of them. Therefore, each primer presented a different picture of the niche overlap among the three horseshoe bats due to their taxonomic affinity.


Assuntos
Quirópteros/fisiologia , Código de Barras de DNA Taxonômico/métodos , Dieta , Genes de Insetos , Metagenoma , Metagenômica/métodos , Animais , Besouros/genética , Primers do DNA/química , Primers do DNA/genética , Dípteros/genética , Cadeia Alimentar , Lepidópteros/genética
11.
Appl Biochem Biotechnol ; 189(4): 1318-1326, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31264104

RESUMO

Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.


Assuntos
Clonagem Molecular , Primers do DNA/química , Mutagênese Sítio-Dirigida/métodos , Reação em Cadeia da Polimerase , Primers do DNA/genética , Vetores Genéticos/química , Vetores Genéticos/genética
12.
Biosens Bioelectron ; 141: 111409, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31207569

RESUMO

The steady increase in commercialization of genetically modified organisms (GMOs) demands low-cost, rapid and portable GMO-detection methods that are technically and economically sustainable. Traditional nucleic acid detection platforms are still expensive, immobile and generate complex read-outs to be analyzed by experienced personal. Herein, we report the development of a portable, rapid and user-friendly GMO-detection biosensor, DaimonDNA. The system specifically amplifies the target DNA using loop-mediated isothermal amplification (LAMP) and provides real-time, naked-eye detection with Hydroxynaphthol blue reagent in less than 30 min. The construction of the platform relies on 3D printing and off-the-shelf electronic components that makes it extremely low-cost (<25 Euro), light weight (108 g), mobile (6 × 6 × 3 cm) and suitable for field deployment. We present the detection of the soybean lectin gene as a species control, and P35S as a transgene element found in many GMO varieties. We confirmed specificity of the DaimonDNA biosensor using" RoundUp Ready (RRS)" and MON89788 soybean genomic DNA with P35S and lectin primer sets. We characterized sensitivity of our system using 76.92, 769.2 and 7692 copies of RRS soybean genomic DNA in a non-GMO background. We benchmarked the DNA amplification and detection efficiency of our system against a thermocycling machine by quantifying the images obtained from gel electrophoresis and showed that our system is comparable to most other reported isothermal amplification techniques. This system can also be used for widespread point-of-care or field-based testing that is infrequently performed due to the lack of rapid, inexpensive, user-friendly and portable methods.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Soja/genética , Colorimetria/instrumentação , Primers do DNA/química , Primers do DNA/genética , DNA de Plantas/análise , Desenho de Equipamento , Naftalenossulfonatos/análise , Impressão Tridimensional , Transgenes
13.
Anal Bioanal Chem ; 411(17): 3871-3880, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31209551

RESUMO

Polymerase chain reaction (PCR) is a powerful technique for the detection and quantification of nucleic acids and has enormous applications to research in molecular biology. Certain inherited diseases, caused by single nucleotide mutations, however, are difficult to identify by PCR, using DNA primers and probes, in a situation where a false diagnosis may lead to incorrect or delayed treatment. With the aim of enhancing the specificity of PCR, we used novel chemically synthesized oligonucleotides containing site-specific methyl phosphotriester (MPTE) inter-nucleoside linkage(s) as primers and probes. The methyl phosphotriester linkages carry no charge, so the reduction in the electrostatic repulsion of an MPTE-DNA/DNA duplex shows stronger hybridization affinity compared to a DNA/DNA duplex. However, the electrosteric effects introduced by the methyl group may result in instability of the double-stranded DNA (dsDNA) formed. With the use of specific MPTE modification sites and optimization of the number of MPTE modifications, greater delta melting temperature (ΔTm) may be obtained, in concert with adjustment of PCR operating conditions, especially with respect to the annealing temperature, to achieve more discriminatory results between the target template and the perfectly matched primer and the mismatched primer. In single nucleotide polymorphism (SNP) genotyping, the results demonstrated that MPTE-modified probes can improve specificity. In summary, MPTE-modified oligonucleotides are a promising DNA analog applied to PCR primers and probes to enhance the specificity and to provide more precise detection results for various applications, such as for genetic diagnosis. In summary, two common DNA polymerases we tested could successfully recognize the MPTE-modified primers and probes. Under the optimal operating conditions, MPTE modification has the ability to improve the discrimination of single nucleotide polymorphism by increasing the ΔTm of the perfect match and mismatch sequences and to provide more precise detection results for various applications, such as genetic diagnosis.


Assuntos
Primers do DNA/química , Fosfatos/química , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sondas de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Genótipo , Metilação , Técnicas de Diagnóstico Molecular
14.
Bioconjug Chem ; 30(6): 1773-1780, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31117344

RESUMO

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.


Assuntos
Primers do DNA/química , Nucleotídeos de Desoxiuracil/química , Desoxiuridina/análogos & derivados , Triazinas/química , Tubercidina/análogos & derivados , Reação de Cicloadição , Primers do DNA/síntese química , DNA Polimerase Dirigida por DNA/química , Nucleotídeos de Desoxiuracil/síntese química , Reação em Cadeia da Polimerase , Triazinas/síntese química , Tubercidina/síntese química , Tubercidina/química
15.
Methods Mol Biol ; 1973: 281-297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016709

RESUMO

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G8AE-clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.


Assuntos
Primers do DNA/química , Oligonucleotídeos/química , RNA de Cadeia Dupla/análise , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética
16.
Methods Mol Biol ; 1973: 299-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016710

RESUMO

Polymerase enzymes catalyze the replication of DNA by incorporating deoxynucleoside monophosphates (dNMPs) into a primer strand in a 5' to 3' direction. Monitoring kinetic aspects of this catalytic process provides mechanistic information regarding polymerase-mediated DNA synthesis and the influences of nucleobase structure. For example, a range of polymerases have different capacities to synthesize DNA depending on the structure of the inserted dNMP (natural or synthetic) and also depending on the templating DNA base (modified vs. unmodified). Under steady-state conditions, relative rates depend on the deoxynucleoside triphosphate (dNTP) residence times in the ternary (polymerase-DNA-dNTP) complex. This chapter describes a method to measure steady-state incorporation efficiencies by which polymerase enzymes insert dNMPs into primer-template (P/T) oligonucleotides. The method described involves the use of a primer oligonucleotide 5' radiolabeled with [γ-32P]ATP. Significant established applications of this experiment include studies regarding mechanisms of nucleotide misincorporation as a basis of chemically induced DNA mutation. Further, it can provide information important in various contexts ranging from biophysical to medical-based studies.


Assuntos
Primers do DNA/química , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos/química , Cinética , Moldes Genéticos
17.
J Biol Chem ; 294(15): 6073-6081, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30842261

RESUMO

Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Linhagem Celular , Cristalografia por Raios X , Primers do DNA/química , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
18.
Vision Res ; 158: 109-119, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30825468

RESUMO

Most diurnal birds have cone-dominated retinae and tetrachromatic colour vision based on ultra-violet/violet-sensitive UV/V cones expressing short wavelength-sensitive opsin 1 (SWS1), S cones expressing short wavelength-sensitive opsin 2 (SWS2), M cones expressing medium wavelength-sensitive opsin (RH2) and L cones expressing long wavelength-sensitive opsin (LWS). Double cones (D) express LWS but do not contribute to colour vision. Each cone is equipped with an oil droplet, transparent in UV/V cones, but pigmented by carotenoids: galloxanthin in S, zeaxanthin in M, astaxanthin in L and a mixture in D cones. Owls (Strigiformes) are crepuscular or nocturnal birds with rod-dominated retinae and optical adaptations for high sensitivity. For eight species, the absence of functional SWS1 opsin has recently been documented, functional RH2 opsin was absent in three of these. Here we confirm the absence of SWS1 transcripts for the Long-eared owl (Asio otus) and demonstrate its absence for the Short-eared owl (Asio flammeus), Tawny owl (Strix aluco) and Boreal owl (Aegolius funereus). All four species had transcripts of RH2, albeit with low expression. All four species express all enzymes needed to produce galloxanthin, but lack CYP2J19 expression required to produce astaxanthin from dietary precursors. We also present ocular media transmittance of the Eurasian eagle owl (Bubo bubo) and Short-eared owl and predict spectral sensitivities of all photoreceptors of the Tawny owl. We conclude that owls, despite lacking UV/V cones, can detect UV light. This increases the sensitivity of their rod vision allowing them, for instance, to see UV-reflecting feathers as brighter signals at night.


Assuntos
Carotenoides/metabolismo , Visão de Cores/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genética , Estrigiformes/fisiologia , Transcriptoma/fisiologia , Raios Ultravioleta , Animais , Primers do DNA/química , Expressão Gênica , Visão Noturna/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Visão Ocular/fisiologia , Xantofilas/metabolismo
19.
J Pharm Biomed Anal ; 169: 181-187, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30877929

RESUMO

A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RCA) had been developed. The aPCR-RCA processes were not new but components of the processes made the assay useful. Twenty-one thymine (21-T) tagged forward primer generated universal twenty-one adenine (21-A) aPCR amplicons after aPCR amplification. A poly-T sequence dumbbell-like RCA template was produced through the blunt-end ligation activity of T4 DNA ligase. After the mixture of aPCR amplicons and dumbbell-like RCA template, the RCA reaction would initiate when the addition of phi29 DNA polymerase, then a large number of G-quadruplex sequences were produced which allowed the intercalation of Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation, THT) for easy fluorescence detection. Under the optimal conditions, the assay showed a limit of detection (LOD) of 4.8 × 101 CFU/mL in pure culture and 4.0 × 102 CFU/g in spiked lettuce homogenates. By changing the aPCR primer, the aPCR-RCA method developed in this study had a potential to detect other bacteria without the design an RCA template for each bacterium.


Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Listeria monocytogenes/química , Reação em Cadeia da Polimerase/métodos , Benzotiazóis/química , Primers do DNA/química , Primers do DNA/genética , Fluorescência , Quadruplex G , Limite de Detecção , Listeria monocytogenes/genética
20.
Curr Protoc Hum Genet ; 101(1): e81, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30620135

RESUMO

Mapping patterns of DNA methylation throughout the epigenome are critical to our understanding of several important biological and regulatory functions, such as transcriptional regulation, genomic imprinting, and embryonic development. The development and rapid advancement of next-generation sequencing (NGS) technologies have provided clinicians and researchers with accurate and reliable read-outs of genomic and epigenomic information at the nucleotide level. Such improvements have significantly lowered the cost required for genome-wide sequencing, facilitating the vast acquisition of data that has led to many improvements in patient care. However, the torrid rate of NGS data generation has left targeted validation approaches behind, including the confirmation of epigenetic marks such as DNA methylation. To overcome these shortcomings, we present a rapid and robust protocol for the parallel examination of multiple methylated sequences that we have termed simultaneous targeted methylation sequencing (sTM-Seq). Key features of this technique include the elimination of the need for large amounts of high-molecular weight DNA and the nucleotide specific distinction of both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Moreover, sTM-Seq is scalable and can be used to investigate multiple loci in dozens of samples within a single sequencing run. By utilizing freely available web-based software and universal primers for multipurpose barcoding, library preparation, and customized sequencing, sTM-Seq is affordable, efficient, and widely applicable. Together, these features enable sTM-Seq to have wide-reaching clinical applications that will greatly improve turnaround rates for same-day procedures and allow clinicians to collect high-resolution data that can be used in a variety of patient settings. © 2019 by John Wiley & Sons, Inc.


Assuntos
Metilação de DNA/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , DNA/genética , Primers do DNA/química , Primers do DNA/genética , Epigênese Genética , Genômica/tendências , Humanos , Software
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