RESUMEN
When the Human Genome Project was completed in 2003, automated Sanger DNA sequencing with fluorescent dye labels was the dominant technology. Several nascent alternative methods based on older ideas that had not been fully developed were the focus of technical researchers and companies. Funding agencies recognized the dynamic nature of technology development and that, beyond the Human Genome Project, there were growing opportunities to deploy DNA sequencing in biological research. Consequently, the National Human Genome Research Institute of the National Institutes of Health created a program-widely known as the Advanced Sequencing Technology Program-that stimulated all stages of development of new DNA sequencing methods, from innovation to advanced manufacturing and production testing, with the goal of reducing the cost of sequencing a human genome first to $100,000 and then to $1,000. The events of this period provide a powerful example of how judicious funding of academic and commercial partners can rapidly advance core technology developments that lead to profound advances across the scientific landscape.
Asunto(s)
Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proyecto Genoma Humano , Análisis de Secuencia de ADN/métodos , HumanosRESUMEN
Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined 'target-capture-PCR' workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost. We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing.
Asunto(s)
Cartilla de ADN/metabolismo , Sondas de ADN/metabolismo , Biblioteca de Genes , Análisis de Secuencia de ADN/métodos , Línea Celular , Variación Genética , Humanos , Mutación INDEL/genética , Polimorfismo de Nucleótido Simple/genética , Estándares de ReferenciaRESUMEN
A challenge in the clinical adoption of cell-free DNA (cfDNA) liquid biopsies for cancer care is their high cost compared to potential reimbursement. The most common approach used in liquid biopsies to achieve high specificity detection of circulating tumor DNA (ctDNA) among a large background of normal cfDNA is to attach molecular barcodes to each DNA template, amplify it, and then sequence it many times to reach a low-error consensus. In applications where the highest possible specificity is required, error rate can be lowered further by independently detecting the sequences of both strands of the starting cfDNA. While effective in error reduction, the additional sequencing redundancy required by such barcoding methods can increase the cost of sequencing up to 100-fold over standard next-generation sequencing (NGS) of equivalent depth. We present a novel library construction and analysis method for NGS that achieves comparable performance to the best barcoding methods, but without the increase in sequencing and subsequent sequencing cost. Named Proximity-Sequencing (Pro-Seq), the method merges multiple copies of each template into a single sequencing read by physically linking the molecular copies so they seed a single sequencing cluster. Since multiple DNA copies of the same template are compared for consensus within the same cluster, sequencing accuracy is improved without the use of redundant reads. Additionally, it is possible to represent both senses of the starting duplex in a single cluster. The resulting workflow is simple, and can be completed by a single technician in a work day with minimal hands on time. Using both cfDNA and cell line DNA, we report the average per-mutation detection threshold and per-base analytical specificity to be 0.003% and >99.9997% respectively, demonstrating that Pro-Seq is among the highest performing liquid biopsy technologies in terms of both sensitivity and specificity, but with greatly reduced sequencing costs compared to existing methods of comparable accuracy.
Asunto(s)
ADN Tumoral Circulante/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Neoplasias/diagnóstico , Línea Celular Tumoral , Humanos , Biopsia Líquida/economía , Neoplasias/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/economíaRESUMEN
Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.
Asunto(s)
Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN/métodos , Mutación , Células Neoplásicas Circulantes/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Fosfatidilinositol 3-Quinasa Clase I/sangre , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN/instrumentación , Predisposición Genética a la Enfermedad , Células HCT116 , Humanos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/patología , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas p21(ras)/sangre , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.
Asunto(s)
ADN Tumoral Circulante/sangre , Neoplasias del Colon/patología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Mutación/genética , Sistema Libre de Células , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/genética , Células Neoplásicas Circulantes/patología , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Circulating tumor DNA (ctDNA) holds promise as a non-invasive means for tumor monitoring in solid malignancies. Assays with high sensitivity and multiplexed analysis of mutations are needed to enable broad application. METHODS: We developed a new assay based on sequence-specific synchronous coefficient of drag alteration (SCODA) technology, which enriches for mutant DNA to achieve high sensitivity and specificity. This assay was applied to plasma and tumor tissue from non-metastatic and metastatic colorectal cancer (CRC) patients, including patients undergoing surgical resection for CRC liver metastases. RESULTS: Across multiple characterization experiments, the assay demonstrated a limit of detection of 0.001% (1 molecule in 100,000) for the majority of the 46 mutations in the panel. In CRC patient samples (n=38), detected mutations were concordant in tissue and plasma for 93% of metastatic patients versus 54% of non-metastatic patients. For three patients, ctDNA identified additional mutations not detected in tumor tissue. In patients undergoing liver metastatectomy, ctDNA anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging. CONCLUSIONS: The multiplexed SCODA mutation enrichment and detection method can be applied to mutation profiling and quantitation of ctDNA, and is likely to have particular utility in the metastatic setting, including patients undergoing metastatectomy.
Asunto(s)
Neoplasias Colorrectales/genética , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/cirugía , ADN de Neoplasias/sangre , Receptores ErbB/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Fosfatidilinositol 3-Quinasas/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras) , Reproducibilidad de los Resultados , Proteínas ras/genéticaRESUMEN
The voltage-driven passage of biological polymers through nanoscale pores is an analytically, technologically, and biologically relevant process. Despite various studies on homopolymer translocation there are still several open questions on the fundamental aspects of pore transport. One of the most important unresolved issues revolves around the passage of biopolymers which vary in charge and volume along their sequence. Here we exploit an experimentally tunable system to disentangle and quantify electrostatic and steric factors. This new, fundamental framework facilitates the understanding of how complex biopolymers are transported through confined space and indicates how their translocation can be slowed down to enable future sensing methods.
Asunto(s)
Biopolímeros/química , ADN/química , Nanoporos , Péptidos/química , Modelos Moleculares , Estructura Molecular , Electricidad EstáticaRESUMEN
Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C)) in the central nervous system into the infectious isoform (PrP(Sc)). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc). A number of pathogenic PrP(C) mutants exist that are characterized by an increased propensity for conversion into PrP(Sc) and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C) conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C), (a PrP(C) mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C) capture event. Moreover, we present a four-state model to describe wild-type PrP(C) kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C). These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.
Asunto(s)
Nanoporos , Enfermedades por Prión/metabolismo , Priones/química , Cinética , Mutación , Priones/genética , Conformación ProteicaRESUMEN
Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.
Asunto(s)
Fagos de Bacillus/enzimología , Fagos de Bacillus/genética , Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Nanoporos , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Dominio Catalítico/fisiología , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/síntesis química , Difosfatos/metabolismo , Activación Enzimática/fisiología , Exonucleasas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Secuencias Invertidas Repetidas/genética , Proteínas Motoras Moleculares/fisiología , Conformación de Ácido NucleicoRESUMEN
Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.
Asunto(s)
Islas de CpG/genética , Citosina/química , Metilación de ADN , ADN/análisis , ADN/genética , Sondas de Oligonucleótidos , ADN/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Mutación/genética , Complejo Represivo Polycomb 2 , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genéticaRESUMEN
A detailed understanding of the kinetics of DNA motion though nanometer-scale pores is important for the successful development of many of the proposed next-generation rapid DNA sequencing and analysis methods. Many of these approaches require DNA motion through nanopores to be slowed by several orders of magnitude from its native translocation velocity so that the translocation times for individual nucleotides fall within practical timescales for detection. With the increased dwell time of DNA in the pore, DNA-pore interactions begin to play an increasingly important role in translocation kinetics. In previous work, we and others observed that when the DNA dwell time in the pore is substantial (>1 ms), DNA motion in α-hemolysin (α-HL) pores leads to nonexponential kinetics in the escape of DNA out of the pore. Here we show that a three-state model for DNA escape, involving stochastic binding interactions of DNA with the pore, accurately reproduces the experimental data. In addition, we investigate the sequence dependence of the DNA escape process and show that the interaction strength of adenine with α-HL is substantially lower relative to cytosine. Our results indicate a difference in the process by which DNA moves through an α-HL nanopore when the motion is fast (microsecond timescale) as compared with when it is slow (millisecond timescale) and strongly influenced by DNA-pore interactions of the kind reported here. We also show the ability of wild-type α-HL to detect and distinguish between 5-methylcytosine and cytosine based on differences in the absolute ionic current through the pore in the presence of these two nucleotides. The results we present here regarding sequence-dependent (and dwell-time-dependent) DNA-pore interaction kinetics will have important implications for the design of methods for DNA analysis through reduced-velocity motion in nanopores.
Asunto(s)
ADN/química , Movimiento (Física) , Nanoestructuras/química , Tamaño de la Partícula , Secuencia de Bases , Simulación por Computador , Electricidad , Proteínas Hemolisinas/química , Iones , Cinética , Modelos Moleculares , Método de Montecarlo , Porosidad , Termodinámica , Factores de TiempoRESUMEN
Methods for the extraction of nucleic acids are straightforward in instances where there is ample nucleic acid mass in the sample and contamination is minimal. However, applications in areas such as metagenomics, life science research, clinical research, and forensics, that are limited by smaller amounts of starting materials or more dilute samples, require sample preparation methods that are more efficient at extracting nucleic acids. Synchronous coefficient of drag alteration (SCODA) is a novel electrophoretic nucleic acid purification technology that has been tested successfully with both highly contaminated and dilute samples and is a promising candidate for new sample preparation challenges. In this article, as an example of SCODA's performance with limited sample material, we outline a genomic DNA (gDNA) extraction protocol from low abundance cultures of Escherichia coli DH10B. This method is equally well suited to high biomass samples.
Asunto(s)
ADN/aislamiento & purificación , Escherichia coli/genética , Genómica/métodosRESUMEN
The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiN(x)) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5-1.0 nm.
Asunto(s)
Nanoestructuras/química , Nanoestructuras/ultraestructura , Proteínas/química , Compuestos de Silicona/química , Difusión , Movimiento (Física) , Tamaño de la Partícula , Porosidad , Conformación Proteica , Pliegue de ProteínaRESUMEN
Weak molecular interactions drive processes at the core of living systems, such as enzyme-substrate interactions, receptor-ligand binding, and nucleic acid replication. Single-molecule force spectroscopy is a remarkable tool for revealing molecular scale energy landscapes of noncovalent bonds, by exerting a mechanical force directly on an individual molecular complex and tracking its survival as a function of time and applied force. In principle, force spectroscopy methods can also be used for highly specific molecular recognition assays, by directly characterizing the strength of bonds between probe and target molecules. However, complexity and low throughput of conventional force spectroscopy techniques render such biosensing applications impractical. Here we demonstrate a straightforward single-molecule approach, suitable for both biophysical studies and molecular recognition assays, in which a approximately 3 nm silicon nitride nanopore is used to determine the bond lifetime spectrum of the biotin-neutravidin complex. Thousands of individual molecular complexes are captured and dissociated in the solid-state nanopore under constant applied forces, ranging from 400 to 900 mV, allowing us to extract the location of the energy barrier that governs the interaction, mapped at Deltax approximately 0.5 nm. These results highlight the capacity of a solid-state nanopore to detect and characterize intermolecular interactions and demonstrate how this could be applied to rapid, highly specific molecular detection assays.
Asunto(s)
Nanotecnología/métodos , Análisis Espectral/métodos , Avidina/metabolismo , Secuencia de Bases , Biotina/metabolismo , ADN/genética , ADN/metabolismo , Ligandos , Datos de Secuencia Molecular , Porosidad , Compuestos de Silicona/química , TermodinámicaRESUMEN
We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands.
Asunto(s)
ADN/aislamiento & purificación , Electroforesis/métodos , Dinámicas no Lineales , Aceites/química , Dióxido de Silicio/químicaRESUMEN
Nanopore analysis of single molecules can be performed by measuring the modulation in ionic current passing through the nanopore while an individual biomolecule such as DNA or RNA is resident in, translocating through, or otherwise interacting with the pore. The corresponding current signature has been shown to reveal properties of the biomolecule and information on its interactions with the pore. The alpha-hemolysin nanopore remains the pore of choice, particularly for single-molecule analysis of nucleic acids, because of its internal dimensions, hydrophilicity, and low-noise characteristics. In this chapter we present a detailed protocol for forming a robust alpha-hemolysin nanopore (or multiple nanopores) for single-molecule analysis.
Asunto(s)
Toxinas Bacterianas/química , Biopolímeros/análisis , Proteínas Hemolisinas/química , Nanoestructuras/química , ADN/análisis , Electrodos , Diseño de Equipo , Membrana Dobles de Lípidos , Nanotecnología , Politetrafluoroetileno , ARN/análisisRESUMEN
Force spectroscopy can be applied using nanopores to study charged molecules such as nucleic acids. This technique can be used to study the binding energy of a DNA duplex by threading an anchored single-stranded DNA (ssDNA) probe molecule through a nanopore (having a diameter large enough to accommodate only a single strand) and allowing target DNA on the backside of the pore to hybridize to the probe. Electric potential can be used to apply a force to the charged ssDNA in a direction tending to translocate the duplex through the pore. If the pore is only large enough to accept ssDNA, the duplex must dissociate for the probe to escape the pore. The dissociation time of the duplex can therefore be measured under applied force, and (provided that enough dissociation events have been recorded) a characteristic time scale for dissociation can be determined. In this chapter, we present a detailed protocol for performing nanopore force spectroscopy on DNA duplexes using one or more alpha-hemolysin nanopores. We present the details of the measurement of the duplex survival probability under force, and show that dissociation time scales for duplexes that are perfectly complimentary differ by greater than approximately two orders of magnitude from those containing a single sequence mismatch, offering opportunities for sequence detection.
Asunto(s)
ADN/química , Nanoestructuras , Análisis Espectral/métodos , ADN/genética , Nanotecnología , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Polimorfismo de Nucleótido Simple , Programas Informáticos , TermodinámicaRESUMEN
A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.
Asunto(s)
Mapeo Cromosómico/tendencias , ADN/genética , Predicción , Nanoestructuras/química , Nanotecnología/tendencias , Alineación de Secuencia/tendencias , Análisis de Secuencia de ADN/tendencias , ADN/química , Genómica/tendencias , Nanoestructuras/ultraestructuraRESUMEN
Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.
Asunto(s)
ADN/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Nanoestructuras/química , ADN/análisis , ADN de Cadena Simple/metabolismo , Cinética , Distribución de Poisson , Porosidad , Electricidad Estática , Procesos Estocásticos , Factores de TiempoRESUMEN
Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for STR profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, leading to frequent failure of STR profiling. We present a novel instrument for DNA purification of forensic samples that is capable of highly effective concentration of nucleic acids from soil particulates, fabric, and other complex samples including solid components. The novel concentration process, known as SCODA, is inherently selective for long charged polymers such as DNA, and therefore is able to effectively reject known contaminants. We present an automated sample preparation instrument based on this process, and preliminary results based on mock forensic samples.