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Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 15 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer mismatches overlapping with primers and predicted PCR byproducts. We also compare Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluate Olivar on real wastewater samples and found that Olivar has up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available online as a web application at https://olivar.rice.edu and can be installed locally as a command line tool with Bioconda. Source code, installation guide, and usage are available at https://github.com/treangenlab/Olivar .
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COVID-19 , Primers do DNA , Genoma Viral , SARS-CoV-2 , SARS-CoV-2/genética , COVID-19/virologia , COVID-19/diagnóstico , Humanos , Genoma Viral/genética , Primers do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Águas Residuárias/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , SoftwareRESUMO
Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 2 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluated Olivar on real wastewater samples and found that Olivar had up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available as a web application at https://olivar.rice.edu. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https://github.com/treangenlab/Olivar.
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One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.
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Algoritmos , Reação em Cadeia da Polimerase Multiplex , Primers do DNA/genética , Funções Verossimilhança , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.
Assuntos
Comunicação Interatrial , Animais , Comunicação Interatrial/genética , Humanos , Camundongos , Proteínas dos Microfilamentos , Mutação/genética , Miofibrilas , Linhagem , Talina , Tropomiosina/genéticaRESUMO
Bicuspid aortic valve (BAV) with ~1%-2% prevalence is the most common congenital heart defect (CHD). It frequently results in valve disease and aorta dilation and is a major cause of adult cardiac surgery. BAV is genetically linked to rare left-heart obstructions (left ventricular outflow tract obstructions [LVOTOs]), including hypoplastic left heart syndrome (HLHS) and coarctation of the aorta (CoA). Mouse and human studies indicate LVOTO is genetically heterogeneous with a complex genetic etiology. Homozygous mutation in the Pcdha protocadherin gene cluster in mice can cause BAV, and also HLHS and other LVOTO phenotypes when accompanied by a second mutation. Here we show two common deletion copy number variants (delCNVs) within the PCDHA gene cluster are associated with LVOTO. Analysis of 1,218 white individuals with LVOTO versus 463 disease-free local control individuals yielded odds ratios (ORs) at 1.47 (95% confidence interval [CI], 1.13-1.92; p = 4.2 × 10-3) for LVOTO, 1.47 (95% CI, 1.10-1.97; p = 0.01) for BAV, 6.13 (95% CI, 2.75-13.7; p = 9.7 × 10-6) for CoA, and 1.49 (95% CI, 1.07-2.08; p = 0.019) for HLHS. Increased OR was observed for all LVOTO phenotypes in homozygous or compound heterozygous PCDHA delCNV genotype comparison versus wild type. Analysis of an independent white cohort (381 affected individuals, 1,352 control individuals) replicated the PCDHA delCNV association with LVOTO. Generalizability of these findings is suggested by similar observations in Black and Chinese individuals with LVOTO. Analysis of Pcdha mutant mice showed reduced PCDHA expression at regions of cell-cell contact in aortic smooth muscle and cushion mesenchyme, suggesting potential mechanisms for BAV pathogenesis and aortopathy. Together, these findings indicate common variants causing PCDHA deficiency play a significant role in the genetic etiology of common and rare LVOTO-CHD.
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Targeted high-throughput DNA sequencing is a primary approach for genomics and molecular diagnostics, and more recently as a readout for DNA information storage. Oligonucleotide probes used to enrich gene loci of interest have different hybridization kinetics, resulting in non-uniform coverage that increases sequencing costs and decreases sequencing sensitivities. Here, we present a deep learning model (DLM) for predicting Next-Generation Sequencing (NGS) depth from DNA probe sequences. Our DLM includes a bidirectional recurrent neural network that takes as input both DNA nucleotide identities as well as the calculated probability of the nucleotide being unpaired. We apply our DLM to three different NGS panels: a 39,145-plex panel for human single nucleotide polymorphisms (SNP), a 2000-plex panel for human long non-coding RNA (lncRNA), and a 7373-plex panel targeting non-human sequences for DNA information storage. In cross-validation, our DLM predicts sequencing depth to within a factor of 3 with 93% accuracy for the SNP panel, and 99% accuracy for the non-human panel. In independent testing, the DLM predicts the lncRNA panel with 89% accuracy when trained on the SNP panel. The same model is also effective at predicting the measured single-plex kinetic rate constants of DNA hybridization and strand displacement.
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Sequência de Bases , Aprendizado Profundo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA/genética , Sondas de DNA , Genômica , Humanos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodosRESUMO
The C5-dicarboxylate α-ketoglutarate (α-KG) is a preferred nutrient source for the opportunistic pathogen Pseudomonas aeruginosa. However, very little is known about how P. aeruginosa detects and responds to α-KG in the environment. Our laboratory has previously shown that the MifS/MifR two-component signal transduction system regulates α-KG assimilation in P. aeruginosa PAO1. In an effort to better understand how this bacterium detects α-KG, we characterized the MifS sensor histidine kinase. In this study we show that although MifS is a homologue of the C4-dicarboxylate sensor DctB, it specifically responds to the C5-dicarboxylate α-KG. MifS activity increased >10-fold in the presence of α-KG, while the related C5-dicarboxylate glutarate caused only a 2-fold increase in activity. All other dicarboxylates tested did not show any significant effect on MifS activity. Homology modelling of the MifS sensor domain revealed a substrate binding pocket for α-KG. Using protein modelling and mutational analysis, we identified nine residues that are important for α-KG response, including one residue that determines the substrate specificity of MifS. Further, we found that MifS has a novel cytoplasmic linker domain that is required for α-KG response and is probably involved in signal transduction from the sensor domain to the cytoplasmic transmitter domain. Until this study, DctB family histidine kinases were known to only respond to C4-dicarboxylates. Our work shows that MifS is a novel member of the DctB family histidine kinase that specifically responds to α-KG.
Assuntos
Histidina Quinase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Pseudomonas aeruginosa/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Ácidos Dicarboxílicos/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/química , Histidina Quinase/genética , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética , Transdução de Sinais , Especificidade por Substrato , Ácido Succínico/metabolismoAssuntos
Nanotecnologia , Neoplasias/diagnóstico , Nanomedicina Teranóstica , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/normas , Análise Mutacional de DNA/métodos , Humanos , Mutação , Nanotecnologia/métodos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Nanomedicina Teranóstica/métodos , Nanomedicina Teranóstica/normasRESUMO
Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock-nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock-nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.
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Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.
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Técnicas Biossensoriais , Proteínas Luminescentes/química , Tiorredoxinas/química , Animais , Linhagem Celular , Glutationa/química , Células HEK293 , Humanos , Oxirredução , Proteína Vermelha FluorescenteRESUMO
Protein structure prediction and modeling provide a tool for understanding protein functions by computationally constructing protein structures from amino acid sequences and analyzing them. With help from protein prediction tools and web servers, users can obtain the three-dimensional protein structure models and gain knowledge of functions from the proteins. In this chapter, we will provide several examples of such studies. As an example, structure modeling methods were used to investigate the relation between mutation-caused misfolding of protein and human diseases including epilepsy and leukemia. Protein structure prediction and modeling were also applied in nucleotide-gated channels and their interaction interfaces to investigate their roles in brain and heart cells. In molecular mechanism studies of plants, rice salinity tolerance mechanism was studied via structure modeling on crucial proteins identified by systems biology analysis; trait-associated protein-protein interactions were modeled, which sheds some light on the roles of mutations in soybean oil/protein content. In the age of precision medicine, we believe protein structure prediction and modeling will play more and more important roles in investigating biomedical mechanism of diseases and drug design.
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Encéfalo/metabolismo , Biologia Computacional/métodos , Epilepsia/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Medicina de Precisão/métodos , Sequência de Aminoácidos , Encéfalo/patologia , Caspase 9/química , Caspase 9/genética , Caspase 9/metabolismo , Caveolina 3/química , Caveolina 3/genética , Caveolina 3/metabolismo , Epilepsia/genética , Epilepsia/patologia , Estudo de Associação Genômica Ampla , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ligantes , Oryza/genética , Melhoramento Vegetal , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de GABA-A/química , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Alinhamento de Sequência , SoftwareRESUMO
[This corrects the article DOI: 10.1128/mSphere.00020-16.].
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Glycine serves as a major source of single carbon units for biochemical reactions within bacterial cells. Utilization of glycine is tightly regulated and revolves around a key group of proteins known as the glycine cleavage system (GCS). Our lab previously identified the transcriptional regulator GcsR (PA2449) as being required for catabolism of glycine in the opportunistic pathogen Pseudomonas aeruginosa PAO1. In an effort to clarify and have an overall better understanding of the role of GcsR in glycine metabolism, a combination of transcriptome sequencing and electrophoretic mobility shift assays was used to identify target genes of this transcriptional regulator. It was found that GcsR binds to an 18-bp consensus sequence (TGTAACG-N4-CGTTCCG) upstream of the gcs2 operon, consisting of the gcvH2, gcvP2, glyA2, sdaA, and gcvT2 genes. The proteins encoded by these genes, namely, the GCS (GcvH2-GcvP2-GcvT2), serine hydroxymethyltransferase (GlyA2), and serine dehydratase (SdaA), form a metabolic pathway for the conversion of glycine into pyruvate, which can enter the central metabolism. GcsR activates transcription of the gcs2 operon in response to glycine. Interestingly, GcsR belongs to a family of transcriptional regulators known as TyrR-like enhancer-binding proteins (EBPs). Until this study, TyrR-like EBPs were only known to function in regulating aromatic amino acid metabolism. GcsR is the founding member of a new class of TyrR-like EBPs that function in the regulation of glycine metabolism. Indeed, homologs of GcsR and its target genes are present in almost all sequenced genomes of the Pseudomonadales order, suggesting that this genetic regulatory mechanism is a common theme for pseudomonads. IMPORTANCE Glycine is required for various cellular functions, including cell wall synthesis, protein synthesis, and the biosynthesis of several important metabolites. Regulating levels of glycine metabolism allows P. aeruginosa to maintain the metabolic flux of glycine through several pathways, including the metabolism of glycine to produce other amino acids, entry into the trichloroacetic acid cycle, and the production of virulence factors such as hydrogen cyanide. In this study, we characterized GcsR, a transcriptional regulator that activates the expression of genes involved in P. aeruginosa PAO1 glycine metabolism. Our work reveals that GcsR is the founding member of a novel class of TyrR-like EBPs that likely regulate glycine metabolism in Pseudomonadales.
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Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl-tRNA synthetase (mbPylRS) to genetically encode a DNP-containing unnatural amino acid, N(6) -(2-(2,4-dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti-DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.
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Aminoacil-tRNA Sintetases/genética , Dinitrobenzenos/química , Haptenos/química , Haptenos/genética , Lisina/análogos & derivados , Methanosarcina barkeri/enzimologia , Dinitrobenzenos/imunologia , Código Genético , Engenharia Genética , Células HEK293 , Haptenos/imunologia , Humanos , Lisina/química , Lisina/genética , Lisina/imunologia , Methanosarcina barkeri/genéticaRESUMO
Catastrophic antiphospholipid antibody syndrome (CAPS) is characterized by fulminant thrombosis of the arterial and venous beds of multiple organ systems over a relatively short period of time and with a high mortality rate. Mucosa-associated lymphoid tissue (MALT) lymphoma of the lung has never been reported as a causative or precipitating factor for CAPS in the CAPS registry database. The present study describes a rare case of pulmonary MALT lymphoma of the lung that presented as CAPS. A 19-year-old Hispanic female presented with shortness of breath and abdominal pain. Computed tomography (CT) scans of the chest and abdomen revealed multiple portal vein thromboses and bilateral pulmonary nodules. Within one week of presentation, the patient developed a straight sinus thrombosis and upper extremity deep vein thrombosis, which led to shortness of breath. A biopsy of the lung nodule revealed MALT lymphoma. The present case illustrates a rarely reported pulmonary MALT lymphoma presenting as CAPS in a young female. The patient was successfully treated with 90 mg/m2 bendamustine on days one and two and rituximab 375 mg/m2 on day one of each 28-day cycle. Complete remission of the lung nodules was observed following three cycles of treatment, as visualized by positron emission tomography (PET)/CT scan. Fondaparinux was identified as a feasible anticoagulation drug of choice for this case. At seven months post-treatment, the patient continues to be stable with no further evidence of thrombosis and is currently undergoing rituximab maintenance therapy every six months for two years. A repeat lupus anticoagulant antibody assay turned and remained negative during the clinical follow-up period. A prompt diagnosis and early aggressive treatment is potentially curative and may dramatically decrease the mortality risk. Future studies should explore the role of rituximab in the management of CAPS-associated B-cell lymphoid malignancies.
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DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg²âº) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg²âº binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5'-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.
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Metilação de DNA , DNA/genética , DNA/metabolismo , Loci Gênicos , Nanoporos , Pareamento de Bases , Técnicas Biossensoriais , Ilhas de CpG , Citosina , UracilaRESUMO
miRNAs are short noncoding RNA molecules that are important in regulating gene expression. Due to the correlation of their expression levels and various diseases, miRNAs are being investigated as potential biomarkers for molecular diagnostics. The fast-growing miRNA exploration demands rapid, accurate, low-cost miRNA detection technologies. This article will focus on two platforms of nanopore single-molecule approach that can quantitatively measure miRNA levels in samples from tissue and cancer patient plasma. Both nanopore methods are sensitive and specific, and do not need labeling, enzymatic reaction or amplification. In the next 5 years, the nanopore-based miRNA techniques will be improved and validated for noninvasive and early diagnosis of diseases.
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MicroRNAs/análise , Nanoporos , Nanotecnologia/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , HumanosRESUMO
A diagnosis of lung cancer at its early stages is vital for improving the survival rate of patients. MicroRNAs (miRNAs), a family of 19- to 25-nucleotide non-coding small RNAs, are frequently dysregulated in lung cancer. The objective of this study was to investigate the potential of circulating miRNAs for early detection of lung cancer. We searched the published literature for the miRNA microarray data of primary lung cancer and selected 15 miRNAs that were most frequently up-regulated in lung cancer tissues. Total plasma RNA including miRNAs was isolated, polyadenylated and reverse-transcribed into cDNAs. The levels of miRNAs were determined by real-time RT-PCR in 74 lung cancer patients and 68 age-matched cancer-free controls. We found that the levels of miR-155, miR-197, and miR-182 in the plasma of lung cancer including stage I patients were significantly elevated compared with controls (P<0.001). The combination of these 3 miRNAs yielded 81.33% sensitivity and 86.76% specificity in discriminating lung cancer patients from controls. The levels of miR-155 and miR-197 were higher in the plasma from lung cancer patients with metastasis than in those without metastasis (P<0.05) and were significantly decreased in responsive patients during chemotherapy (P<0.001). These results indicate that miR-155, miR-197, and miR-182 can be potential non-invasive biomarkers for early detection of lung cancer.
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Carcinoma Broncogênico/diagnóstico , Neoplasias Pulmonares/diagnóstico , MicroRNAs/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Carcinoma Broncogênico/sangue , Carcinoma Broncogênico/secundário , Diagnóstico Precoce , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MicroRNAs are short RNA molecules that regulate gene expression, and have been investigated as potential biomarkers because their expression levels are correlated with various diseases. However, detecting microRNAs in the bloodstream remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor based on the α-haemolysin protein can selectively detect microRNAs at the single molecular level in plasma samples from lung cancer patients without the need for labels or amplification of the microRNA. The sensor, which uses a programmable oligonucleotide probe to generate a target-specific signature signal, can quantify subpicomolar levels of cancer-associated microRNAs and can distinguish single-nucleotide differences between microRNA family members. This approach is potentially useful for quantitative microRNA detection, the discovery of disease markers and non-invasive early diagnosis of cancer.
Assuntos
Neoplasias Pulmonares/sangue , MicroRNAs/sangue , Nanoporos , Sondas de Oligonucleotídeos/química , RNA Neoplásico/sangue , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , MasculinoRESUMO
A novel, easy to perform PCR-based method employing specific DNA methylation biomarkers to detect B-cell neoplasms in a variety of B-cell lines and B lymphoblastic leukemia (B-ALL) patient specimens has been developed. This method detects as few as 5 B-ALL cells, or 1 B-ALL cell in 1,000,000 normal background blood cells using a single marker, DLC-1 gene CpG island (CGI) methylation. By adding two additional markers PCDHGA12 and RPIB9, over 80% of B-ALL cases were detected in patients' bone marrow and/or peripheral blood specimens. We have traced clinical B-ALL cases up to 10 years retrospectively and the DLC-1 methylation is correlated with patient clinical status. Thus, this epigenetic-based molecular method demonstrates its potential use in the diagnosis of B-cell neoplasia, in addition to traditional approach such as clinical features, morphology, immunophenotype, and genetic analysis.