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1.
Talanta ; 236: 122846, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635236

RESUMO

Simultaneous detection of multiple microRNAs (miRNAs) with high sensitivity can give accurate and reliable information for clinical applications. By uniformly anchoring hairpin probes on the surface of DNA nanolantern, a three-dimensional DNA nanostructure contains abundant and adjustable modification sites, highly integrated DNA nanoprobes were designed and developed as catalytic hairpin assembly (CHA)-based signal amplifiers for enzyme-free signal amplification detection of target miRNAs. The nanolantern-based CHA (NLC) amplifiers, which were facilely prepared via a simple "one-pot" annealing method, showed enhanced biostability, improved cell internalization efficiency, accelerated CHA reaction kinetics, and increased signal amplification capability compared to the single-stranded DNA hairpin probes used in traditional CHA reaction. By co-assembling multiple hairpin probes on a DNA nanolantern surface, as-prepared NLC amplifiers were demonstrated to work well for highly sensitive and specific imaging, expression level fluctuation analysis of two miRNAs in living cells, and miRNAs-guided tumor imaging in living mice. The proposed DNA nanolantern-based nanoamplifier strategy might provide a feasible way to promote the cellular and in vivo applications of nucleic acid probes.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Animais , Catálise , DNA/genética , Camundongos , MicroRNAs/genética , Sondas de Ácido Nucleico
2.
Anal Chem ; 93(35): 12096-12102, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34432421

RESUMO

Developing spherical nucleic acids with new structures holds great promise for nanomedicine and bioanalytical fields. Covalent organic frameworks (COFs) are emerging promising materials with unique properties for a wide range of applications. However, devising COF-based spherical nucleic acid is challenging because methods for the preparation of functionalized COFs are still limited. We report here a bonding defect-amplified modification (BDAM) strategy for the facile preparation of functionalized COFs. Poly(acrylic acid) was employed as the defect amplifier to modify the surface of COF nanoparticles by the formation of amide bonds with amino residues, which successfully converted and amplified the residues into abundant reactive carboxyl groups. Then, amino terminal-decorated hairpin DNA was densely grafted onto the surface of COF nanoparticles (NPs) to give rise to a spherical nucleic acid probe (SNAP). A series of experiments and characterizations proved the successful preparation of the COF-based SNAP, and its application in specifically lighting up RNA biomarkers in living cells for cancer diagnostic imaging was demonstrated. Therefore, the COF-based SNAP is a promising candidate for biomedical applications and the proposed BDAM represents a useful strategy for the preparation of functionalized COFs for diverse fields.


Assuntos
Estruturas Metalorgânicas , Nanopartículas , Ácidos Nucleicos , DNA/genética , Sondas de Ácido Nucleico
3.
Biochem Biophys Res Commun ; 569: 174-178, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34252589

RESUMO

Adenosine deaminase-dependent RNA editing is a widespread universal mechanism of posttranscriptional gene function modulation. Changes in RNA editing level may contribute to various physiological and pathological processes. In the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor GluA2 subunit, A-I editing in the Q607R site leads to dramatic changes in function, making the receptor channel calcium-impermeable. A standard approach for quantifying (un)edited RNAs is based on endpoint PCR (Sanger sequencing or restriction analysis), a time-consuming and semiquantitative method. We aimed to develop RT-qPCR assays to quantify rat Q607R (A-I) edited/unedited mRNA in samples in the present work. Based on self-probing PCR detection chemistry, described initially for detecting short DNA fragments, we designed and optimised RT-qPCR assays to quantify Q607R (un)edited mRNA. We used self-probing primer PCR technology for mRNA quantification for the first time. Using a novel assay, we confirmed that Q607R GluA2 mRNA editing was increased in 14-day- (P14) or 21-day-old (P21) postnatal brain tissue (hippocampus) compared to the embryonic brain (whole brains at E20) in Wistar rats. Q607R unedited GluA2 mRNA was detectable by our assay in the cDNA of mature brain tissue compared to that derived through classical methods. Thus, self-probing primer PCR detection chemistry is an easy-to-use approach for RT-qPCR analysis of RNA editing.


Assuntos
Expressão Gênica , Hipocampo/metabolismo , Edição de RNA , RNA Mensageiro/genética , Receptores de AMPA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Masculino , Sondas de Ácido Nucleico/genética , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes , Fatores de Tempo
4.
Genome Biol ; 22(1): 169, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082799

RESUMO

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.


Assuntos
COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sondas de Ácido Nucleico/genética , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e Especificidade
5.
Methods Mol Biol ; 2281: 209-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847960

RESUMO

Fluorescent in situ hybridization coupled with immunofluorescence (FISH/IF) is an assay that has been widely used to study DNA-protein interactions. The technique is based on the use of a fluorescent nucleic acid probe and fluorescent antibodies to reveal the localization of a DNA sequence and a specific protein in the cell. The interaction can be inferred by the quantification of the co-localization between the protein and the DNA. Here, we describe a detailed FISH/IF methodology that our group used to study RPA-telomere interaction in the pathogenic protozoa parasite Trypanosoma cruzi.


Assuntos
Proteína de Replicação A/metabolismo , Telômero/metabolismo , Trypanosoma cruzi/metabolismo , Imunofluorescência , Hibridização in Situ Fluorescente , Sondas de Ácido Nucleico/química , Proteínas de Protozoários/metabolismo , Telômero/química , Trypanosoma cruzi/genética
6.
ACS Sens ; 6(5): 1949-1955, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33905657

RESUMO

Nucleic acids as the important tumor markers play a crucial role in the identification of cancer. Various kinds of probes such as gold nanoparticles and graphene oxide have been explored to detect different nucleic acid markers. However, the existing probes are mostly used to detect a single tumor marker and susceptible to harsh conditions in the complex and dynamic physiological environment, which may lead to false positive results and greatly limit the sensing performance of the probe. Herein, a powerful and reliable Au-Se probe was developed for high-fidelity imaging of two cancer markers simultaneously in living cells. Compared with the traditional nucleic acid probe based on the Au-S bond, this probe was more stable against biological thiols and could effectively distinguish normal cells and cancer cells to avoid false positive results, which is more suitable for imaging in a complex physiological environment. This strategy will provide more valuable insights into designing and exploring novel biosensors in the future.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Ouro , Sondas de Ácido Nucleico , Compostos de Sulfidrila
7.
Methods Mol Biol ; 2246: 1-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576979

RESUMO

Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification, and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms and, the clinical, for the rapid diagnostics of infectious agents. The FISH method is based on the hybridization of a fluorescently labeled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, an hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell. Here, we will review the major steps involved in a standard FISH protocol, namely, fixation/permeabilization, hybridization, washing, and visualization/detection. For each step, the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail.


Assuntos
Hibridização in Situ Fluorescente/métodos , Microbiota/genética , Fluorescência , Sondas de Ácido Nucleico/genética , Sondas de Oligonucleotídeos/genética , RNA Bacteriano/genética , RNA Ribossômico/genética
8.
Methods Mol Biol ; 2246: 69-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576983

RESUMO

Traditionally, RNA and DNA probes are used in fluorescence in situ hybridization (FISH) methods for microbial detection and characterization of communities' structure and diversity. However, the recent introduction of nucleic acid mimics (NAMs) has improved the robustness of the FISH methods in terms of sensitivity and specificity. Several NAMs have been used, of which the most relevant are peptide nucleic acid (PNA), locked nucleic acids (LNA), 2'-O-methyl RNA (2'OMe), and phosphorothioates (PS). In this chapter, we describe a protocol using PNA and LNA/2'OMe probes for microbial detection by FISH, pointing out the differences between them. These protocols are easily adapted to different microorganisms and different probe sequences.


Assuntos
Hibridização in Situ Fluorescente/métodos , Ácidos Nucleicos/genética , Microbiota/genética , Sondas de Ácido Nucleico/genética , Oligonucleotídeos/genética , Ácidos Nucleicos Peptídicos/genética , Sensibilidade e Especificidade
9.
Methods Mol Biol ; 2246: 263-277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576995

RESUMO

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.


Assuntos
Bactérias/genética , Hibridização in Situ Fluorescente/métodos , Sondas de Ácido Nucleico/genética , Ácidos Nucleicos/genética , Oligonucleotídeos/genética
10.
J Microbiol Biotechnol ; 31(3): 358-367, 2021 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-33397829

RESUMO

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.


Assuntos
COVID-19/diagnóstico , Sondas de Ácido Nucleico/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Animais , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Dosagem de Genes/genética , Humanos , RNA Viral/genética , Sensibilidade e Especificidade , Células Vero
11.
Chem Biol Drug Des ; 97(4): 865-892, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33314595

RESUMO

Peptide nucleic acids (PNAs) are getting prodigious interest currently in the biomedical and diagnostic field as an extremely powerful tool because of their potentiality to hybridize with natural nucleic acids. Although PNA has strong affinity and sequence specificity to DNA/RNA, there is a considerable ongoing effort to further enhance their special chemical and biological properties for potential application in numerous fields, notably in the field of therapeutics. The toolbox for backbone modified PNAs synthesis has been extended substantially in recent decades, providing a more efficient synthesis of peptides with numerous scaffolds and modifications. This paper reviews the various strategies that have been developed so far for the modification of the PNA backbone, challenging the search for new PNA systems with improved chemical and physical properties lacking in the original aegPNA backbone. The various practical issues and limitations of different PNA systems are also summarized. The focus of this review is on the evolution of PNA by its backbone modification to improve the cellular uptake, sequence specificity, and compatibility of PNA to bind to DNA/RNA. Finally, an insight was also gained into major applications of backbone modified PNAs for the development of biosensors.


Assuntos
Evolução Molecular , Ácidos Nucleicos Peptídicos/química , Técnicas Biossensoriais/métodos , DNA/química , DNA/metabolismo , Sondas de Ácido Nucleico/química , Sondas de Ácido Nucleico/metabolismo , Ácidos Nucleicos Peptídicos/metabolismo , RNA/química , RNA/metabolismo , Estereoisomerismo
12.
Biotechnol Bioeng ; 117(10): 3212-3223, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32946120

RESUMO

Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 102 - 4.39 × 103 s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.


Assuntos
Corantes Fluorescentes/química , Hibridização in Situ Fluorescente/métodos , Sondas de Ácido Nucleico/química , Animais , Bactérias , Células Cultivadas , Difusão
13.
Biosensors (Basel) ; 10(9)2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32906640

RESUMO

A carbon nanofiber enriched 8-channel screen-printed electrochemical array was used for the impedimetric detection of SNP related to Factor V Leiden (FV Leiden) mutation, which is the most common inherited form of thrombophilia. FV Leiden mutation sensing was carried out in three steps: solution-phase nucleic acid hybridization between zip nucleic acid probe (Z-probe) and mutant type DNA target, followed by the immobilization of the hybrid on the working electrode area of array, and measurement by electrochemical impedance spectroscopy (EIS). TArzhe selectivity of the assay was tested against mutation-free DNA sequences and synthetic polymerase chain reaction (PCR) samples. The developed biosensor was a trustful assay for FV Leiden mutation diagnosis, which can effectively discriminate wild type and mutant type even in PCR samples.


Assuntos
Técnicas Biossensoriais , Fator V/análise , Sondas de Ácido Nucleico , Espectroscopia Dielétrica , Eletrodos , Mutação , Nanofibras , Hibridização de Ácido Nucleico , Trombofilia
14.
Sensors (Basel) ; 20(14)2020 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-32664471

RESUMO

Application of restriction endonuclease (REase) enzymes for specific detection of nucleic acids provides for high assay specificity, convenience and low cost. A direct restriction assay format is based on the specific enzymatic cleavage of a target-probe hybrid that is accompanied with the release of a molecular marker into the solution, enabling target quantification. This format has the detection limit in nanomolar range. The assay sensitivity is improved drastically to the attomolar level by implementation of exponential signal amplification that is based on a cascade of self-perpetuating restriction endonuclease reactions. The cascade is started by action of an amplification "trigger". The trigger is immobilized through a target-specific probe. Upon the target probe hybridization followed with specific cleavage, the trigger is released into the reaction solution. The solution is then added to the assay amplification stage, and the free trigger induces cleavage of amplification probes, thus starting the self-perpetuating cascade of REase-catalyzed events. Continuous cleavage of new amplification probes leads to the exponential release of new triggers and rapid exponential signal amplification. The proposed formats exemplify a valid isothermal alternative to qPCR with similar sensitivity achieved at a fraction of the associated costs, time and labor. Advantages and challenges of the approach are discussed.


Assuntos
Enzimas de Restrição do DNA , DNA/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Limite de Detecção , Hibridização de Ácido Nucleico , Sondas de Ácido Nucleico
15.
Anal Chem ; 92(12): 8459-8463, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32423196

RESUMO

Spherical nucleic acid probes (SNAPs) are 3D nucleic acid nanostructures with multiple superiorities over bare nucleic acid chains. Au-based SNAPs that employ gold nanoparticles (AuNPs) as cores and densely modified nucleic acid chains (commonly via Au-S bonds) as shells have been extensively investigated for the diagnosis and therapy of diverse diseases. However, abundant biothiols in living cells can severely displace nucleic acid chains from AuNPs and restrict their theranostic performance. Herein we report the design and preparation of a selenol terminal-functionalized molecular beacon (MB-SeH), which was further employed to prepare a Au-Se bond-based SNAP (SNAP-Se) for bioimaging. A series of experiments proved the successful preparation of MB-SeH and SNAP-Se, and the obtained nanoprobe could avoid biothiol interference and eliminate the false positive signals during biomarker imaging in living cells. This work will open a new avenue for the design and application of SNAPs.


Assuntos
Ouro/química , MicroRNAs/análise , Sondas de Ácido Nucleico/química , Compostos de Selênio/química , Citometria de Fluxo , Humanos , Células MCF-7 , Microscopia Confocal , Estrutura Molecular , Imagem Óptica
16.
Mikrochim Acta ; 187(4): 238, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32189135

RESUMO

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.


Assuntos
Colorimetria/métodos , Genótipo , Sondas de Ácido Nucleico/química , Ácidos Nucleicos Peptídicos , Talassemia/genética , Biotinilação , Carboximetilcelulose Sódica , DNA/metabolismo , Humanos , Sondas de Ácido Nucleico/metabolismo , Pigmentação
17.
Methods Mol Biol ; 2105: 217-230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32088873

RESUMO

Fluorescence in situ hybridization (FISH) is a 30-year-old technology that has evolved continuously and is now one of the most well-established molecular biology techniques. Traditionally, DNA probes are used for in situ hybridization. However, synthetic molecules are emerging as very promising alternatives, providing better hybridization performance and making FISH procedures easier and more efficient. In this chapter, we describe a universal FISH protocol, using nucleic acid probes, for the detection of bacteria. This protocol should be easily applied to different microorganisms as a way of identifying in situ relevant microorganisms (including pathogens) and their distribution patterns in different types of samples.


Assuntos
Hibridização in Situ Fluorescente , Técnicas Microbiológicas , Ácidos Nucleicos Peptídicos , Bactérias/genética , Técnicas Bacteriológicas , Biofilmes , Bases de Dados Factuais , Dermoscopia , Humanos , Hibridização in Situ Fluorescente/métodos , Sondas de Ácido Nucleico , RNA Ribossômico 16S
18.
Forensic Sci Int Genet ; 45: 102228, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31911363

RESUMO

Analysis of single nucleotide polymorphisms (SNPs) in mitochondrial (mt)DNA hypervariable regions (HV) 1/2 is valuable in forensic investigations. We developed a method for mtDNA screening of the HV1 and HV2 regions by melting curve analysis, using peptide nucleic acid (PNA) probes. This method focuses on melting peak patterns obtained by thermal dissociation of PNA/DNA duplexes in amplified mtDNA products. Five PNA probe sets were designed to detect 25 SNPs in the two HV regions. We also detected non-target SNPs based on unexpected melting temperature (Tm) shifts. In fact, 62 SNPs (42 SNPs in HV1 and 20 in HV2) were identified, including the 25 target SNPs. Using this method, 46 melting peak patterns, including 8 pattern groups, were obtained in 60 unrelated individuals. The peak patterns were compared to 55 haplotypes identified by Sanger sequencing. The results obtained from analysis of target mtDNA SNPs were entirely consistent with those obtained by Sanger sequencing. Screening the HV1 and HV2 regions of mtDNA by this method may help minimize unnecessary recourse to full sequence analysis, allows to rapidly exclude samples that do not match evidence and reference samples, and may reduce turnaround times and analysis costs. Overall, this method may be effective and helpful in forensic investigations.


Assuntos
DNA Mitocondrial/genética , Sondas de Ácido Nucleico , Ácidos Nucleicos Peptídicos/genética , Polimorfismo de Nucleotídeo Único , Temperatura de Transição , Genética Forense/métodos , Genótipo , Humanos , Técnicas Analíticas Microfluídicas , Reação em Cadeia da Polimerase
19.
Top Curr Chem (Cham) ; 378(1): 10, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31894426

RESUMO

Single-nucleotide variants (SNVs) that are strongly associated with many genetic diseases and tumors are important both biologically and clinically. Detection of SNVs holds great potential for disease diagnosis and prognosis. Recent advances in DNA nanotechnology have offered numerous principles and strategies amenable to the detection and quantification of SNVs with high sensitivity, specificity, and programmability. In this review, we will focus our discussion on emerging techniques making use of DNA strand displacement, a basic building block in dynamic DNA nanotechnology. Based on their operation principles, we classify current SNV detection methods into three main categories, including strategies using toehold-mediated strand displacement reactions, toehold-exchange reactions, and enzyme-mediated strand displacement reactions. These detection methods discriminate SNVs from their wild-type counterparts through subtle differences in thermodynamics, kinetics, or response to enzymatic manipulation. The remarkable programmability of dynamic DNA nanotechnology also allows the predictable design and flexible operation of diverse strand displacement probes and/or primers. Here, we offer a systematic survey of current strategies, with an emphasis on the molecular mechanisms and their applicability to in vitro diagnostics.


Assuntos
DNA/química , DNA/genética , Variação Genética , Nucleotídeos/genética , Humanos , Nanotecnologia , Hibridização de Ácido Nucleico , Sondas de Ácido Nucleico/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
20.
Methods Mol Biol ; 2107: 199-231, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893449

RESUMO

Target enrichment sequencing (TES) is a powerful approach to deep-sequencing the exome or genomic regions of interest with great depth. Although successfully and widely adopted in many plant species, TES is currently applied for genotyping of only a couple legume species. Here we describe an in-solution probe capture based method for application of TES in legumes. The topics cover probe design, library preparation, probe hybridization, as well as bioinformatic analysis for evaluation of target capture efficiency and identifying single nucleotide polymorphisms using generated sequencing data.


Assuntos
Fabaceae/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sondas de Ácido Nucleico/genética , Polimorfismo de Nucleotídeo Único , Biologia Computacional , Exoma , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Técnicas de Genotipagem , Análise de Sequência de DNA/métodos
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