Detection Of Single Point Mutations Research Articles

Potential of Circulating Tumor DNA for the Management of Patients With Lymphoma.

The characterization of circulating tumor-derived DNA (ctDNA) has recently emerged in the field of oncology as a powerful method to identify tumor-specific genetic aberrations using peripheral blood testing. Several technical precautions are needed at the pre-analytic stage (given the short half-life of free nucleic acids in plasma), and numerous techniques-with different sensitivities-are available to identify these molecular aberrations, ranging from the detection of single point mutations to extended genetic screening panels. Although a "liquid biopsy" cannot be substituted for the pathological examination of tissue specimens for diagnostic purposes, it can sometimes complement pathology results or serve as a proxy approach for particular lymphoma presentations where biopsies are sometimes difficult to perform. Moreover, ctDNA testing can characterize, at diagnosis or during treatment, mutations that may contribute to the choice of an optimal targeted therapy (such as Bruton tyrosine kinase or EZH2 inhibitors) or detect the emergence of resistance to those therapies. High levels of ctDNA before treatment appear to be correlated with advanced disease stages and prognosis in diffuse large B-cell and follicular lymphomas. Real-time follow-up of ctDNA levels during therapy in several lymphoma subtypes (diffuse large B-cell and Hodgkin lymphomas) has been explored: preliminary studies have demonstrated that this monitoring technique can predict clinical outcomes (end of treatment response and risk of progression after treatment completion) and that this approach may complement the information provided by metabolic imaging assessments. Technical standardization and careful prospective evaluation of the role of ctDNA monitoring in clinical studies represent current important challenges to allowing its application in routine practice.

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

It has now been established that in biological fluids such as blood, it is possible to detect cancer causing genomic alterations by analysing circulating tumour DNA (ctDNA). Information derived from ctDNA offers a unique opportunity to enrich our understanding of cancer biology, tumour evolution and therapeutic efficacy and resistance. Here, we propose a workflow to identify targeted mutations by a customized microarray-based assay for the simultaneous detection of single point mutations in different oncogenes (KRAS, NRAS and BRAF) followed by droplet digital PCR (ddPCR) to determine the fractional abundance of the mutated allele. Genetic variants were determined in the plasma of 20 metastatic colorectal cancer (mCRC) patients previously genotyped on tissue biopsy at the diagnosis for medication planning (T0) and following the tumour genetic evolution during treatment phase (T1 and T2) with the objective of allowing therapy response prediction and monitoring. Our preliminary results show that this combined approach is suitable for routine clinical practice. The microarray platform enables for a rapid, specific and sensitive detection of the most common mutations suitable for high-throughput analysis without costly instrumentation while, the ddPCR, consents an absolute quantification of the mutated allele in a longitudinal observational study on patients undergoing targeted therapy.

An integrated multi-molecular sensor for simultaneous BRAFV600E protein and DNA single point mutation detection in circulating tumour cells.

The analysis of circulating cancer biomarkers in the form of liquid biopsies confers several potential benefits as compared to traditional surgical tissue sampling. As a common key anomaly strongly implicated across several cancer types, the BRAFV600E mutation is one of the most valuable oncogenic biomarkers available in liquid biopsies. Crucially, BRAFV600E is also an actionable mutation which could be arrested by clinically beneficial drug inhibitors. Yet, as is true for most single base disease mutations, current BRAFV600E detection in either its DNA or protein molecular state is still liable to false positive/negative outcomes, thus impacting patient treatment benefit. Here we present an integrated multi-molecular sensor (IMMS) for an entire sample-to-answer workflow from melanoma cell capture to simultaneous quantification of both intracellular BRAFV600E DNA and protein levels on a single platform. The IMMS combines (i) specific capture and release of circulating melanoma cells; (ii) electric field-induced cell lysis; (iii) simultaneous quantification of BRAFV600E DNA and protein levels. We investigated the IMMS system's analytical performance in cell capture, release and lysis, and intracellular BRAFV600E detection by ligase-mediated DNA amplification and antibody-based protein hybridization. As a proof-of-concept, we successfully demonstrated circulating BRAFV600E detection at both DNA and protein molecular levels in simulated melanoma plasma samples. With its capabilities in integrated and miniaturized analysis, the IMMS could lead the emergence of a new generation of multi-molecular lab-on-chip biosensors for enabling more accurate and extensive analysis of powerful circulating biomarkers in patient liquid biopsies.

Graphene Doped Mn2O3 Nanofibers as a Facile Electroanalytical DNA Point Mutation Detection Platform for Early Diagnosis of Breast/Ovarian Cancer

AbstractThis paper demonstrates a simple, label‐free detection methodology for detecting single point DNA mutations. Single point mutation detection is a key enabler for diagnosis and prevention of several genetic disorders that manifest into cancers. Specifically for this purpose, herein, an electrochemical biosensor utilizing electrospun graphene doped manganese III oxide nanofibers (GMnO) is developed. The charge transfer resistance offered by GMnO is extremely sensitive to the localized change in the conductivity. This sensitivity, attributed to the low band gap of Mn2O3 and high charge transfer kinetics of graphene, is explored in the proposed mutation detection platform. As a proof of concept, ultrasensitive detection of BRCA1 gene specific point mutation is demonstrated. The target specific single stranded probe DNA is immobilized onto GMnO modified glassy carbon working electrodes via chemisorption. Post target‐DNA hybridization, differential pulse voltammetry is employed to facilitate detection of targeted point mutation, wherein, difference in peak currents is used to distinguish the target DNA as normal or mutant. Efficiency of the proposed method is evaluated against a target concentration ranging from 10 pM−1 μM. With respect to the mutated target DNA, the LoD of the proposed device is found to be 0.8±0.069 pM. The proposed approach can be extended for detecting any mutation/hybridization of interest by simply adapting an appropriate functionalization protocol.

Rational design of non-resistant targeted cancer therapies

Drug resistance is one of the major problems in targeted cancer therapy. A major cause of resistance is changes in the amino acids that form the drug-target binding site. Despite of the numerous efforts made to individually understand and overcome these mutations, there is a lack of comprehensive analysis of the mutational landscape that can prospectively estimate drug-resistance mutations. Here we describe and computationally validate a framework that combines the cancer-specific likelihood with the resistance impact to enable the detection of single point mutations with the highest chance to be responsible of resistance to a particular targeted cancer therapy. Moreover, for these treatment-threatening mutations, the model proposes alternative therapies overcoming the resistance. We exemplified the applicability of the model using EGFR-gefitinib treatment for Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Cancer (LSCC) and the ERK2-VTX11e treatment for melanoma and colorectal cancer. Our model correctly identified the phenotype known resistance mutations, including the classic EGFR-T790M and the ERK2-P58L/S/T mutations. Moreover, the model predicted new previously undescribed mutations as potentially responsible of drug resistance. Finally, we provided a map of the predicted sensitivity of alternative ERK2 and EGFR inhibitors, with a particular highlight of two molecules with a low predicted resistance impact.

RNA-TVcurve: a Web server for RNA secondary structure comparison based on a multi-scale similarity of its triple vector curve representation

BackgroundRNAs have been found to carry diverse functionalities in nature. Inferring the similarity between two given RNAs is a fundamental step to understand and interpret their functional relationship. The majority of functional RNAs show conserved secondary structures, rather than sequence conservation. Those algorithms relying on sequence-based features usually have limitations in their prediction performance. Hence, integrating RNA structure features is very critical for RNA analysis. Existing algorithms mainly fall into two categories: alignment-based and alignment-free. The alignment-free algorithms of RNA comparison usually have lower time complexity than alignment-based algorithms.ResultsAn alignment-free RNA comparison algorithm was proposed, in which novel numerical representations RNA-TVcurve (triple vector curve representation) of RNA sequence and corresponding secondary structure features are provided. Then a multi-scale similarity score of two given RNAs was designed based on wavelet decomposition of their numerical representation. In support of RNA mutation and phylogenetic analysis, a web server (RNA-TVcurve) was designed based on this alignment-free RNA comparison algorithm. It provides three functional modules: 1) visualization of numerical representation of RNA secondary structure; 2) detection of single-point mutation based on secondary structure; and 3) comparison of pairwise and multiple RNA secondary structures. The inputs of the web server require RNA primary sequences, while corresponding secondary structures are optional. For the primary sequences alone, the web server can compute the secondary structures using free energy minimization algorithm in terms of RNAfold tool from Vienna RNA package.ConclusionRNA-TVcurve is the first integrated web server, based on an alignment-free method, to deliver a suite of RNA analysis functions, including visualization, mutation analysis and multiple RNAs structure comparison. The comparison results with two popular RNA comparison tools, RNApdist and RNAdistance, showcased that RNA-TVcurve can efficiently capture subtle relationships among RNAs for mutation detection and non-coding RNA classification. All the relevant results were shown in an intuitive graphical manner, and can be freely downloaded from this server. RNA-TVcurve, along with test examples and detailed documents, are available at: http://ml.jlu.edu.cn/tvcurve/.

Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection.

The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia.

Single point mutation detection in living cancer cells by far-red emitting PNA-FIT probes.

Peptide nucleic acid bis-quinoline conjugates are reported as attractive far-red emitting probes that detect mutated mRNA in living cells at SNP resolution.

Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain.

Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10(-12) M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components.

Single-point mutation detection in RNA extracts using gold nanoparticles modified with hydrophobic molecular beacon-like structures

Gold nanoparticles functionalized with oligonucleotides that bear a cholesterol group are used as gene sensors. The hydrophobic molecule is buried inside the nanostructure but when the complementary RNA sequence is present the structure unfolds exposing the cholesterol group to the water molecules. This rearrangement leads to the aggregation of the nanostructures.

Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads® with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acids.

Automated extraction and semantic analysis of mutation impacts from the biomedical literature

BackgroundMutations as sources of evolution have long been the focus of attention in the biomedical literature. Accessing the mutational information and their impacts on protein properties facilitates research in various domains, such as enzymology and pharmacology. However, manually curating the rich and fast growing repository of biomedical literature is expensive and time-consuming. As a solution, text mining approaches have increasingly been deployed in the biomedical domain. While the detection of single-point mutations is well covered by existing systems, challenges still exist in grounding impacts to their respective mutations and recognizing the affected protein properties, in particular kinetic and stability properties together with physical quantities.ResultsWe present an ontology model for mutation impacts, together with a comprehensive text mining system for extracting and analysing mutation impact information from full-text articles. Organisms, as sources of proteins, are extracted to help disambiguation of genes and proteins. Our system then detects mutation series to correctly ground detected impacts using novel heuristics. It also extracts the affected protein properties, in particular kinetic and stability properties, as well as the magnitude of the effects and validates these relations against the domain ontology. The output of our system can be provided in various formats, in particular by populating an OWL-DL ontology, which can then be queried to provide structured information. The performance of the system is evaluated on our manually annotated corpora. In the impact detection task, our system achieves a precision of 70.4%-71.1%, a recall of 71.3%-71.5%, and grounds the detected impacts with an accuracy of 76.5%-77%. The developed system, including resources, evaluation data and end-user and developer documentation is freely available under an open source license at http://www.semanticsoftware.info/open-mutation-miner.ConclusionWe present Open Mutation Miner (OMM), the first comprehensive, fully open-source approach to automatically extract impacts and related relevant information from the biomedical literature. We assessed the performance of our work on manually annotated corpora and the results show the reliability of our approach. The representation of the extracted information into a structured format facilitates knowledge management and aids in database curation and correction. Furthermore, access to the analysis results is provided through multiple interfaces, including web services for automated data integration and desktop-based solutions for end user interactions.

Selective Aggregation Mechanism of Unmodified Gold Nanoparticles in Detection of Single Nucleotide Polymorphism

Detection of single point mutation based on the hybridization of oligonucleotides was performed using unmodified gold nanoparticles. The sequences of oligonucleotides were designed to detect the metastatic efficiency modifier signal-induced proliferation-associated gene 1 (Sipa1). The detection step was monitored using UV−vis absorption spectroscopy, quasielastic light scattering, and zeta potential measurement. We observed that addition of DNAs into the suspension of unmodified gold nanoparticles could substantially aggregate the gold nanoparticles and change the color of solution. By changing the salt concentration in the presence of a phosphate buffer solution, we were able to selectively aggregate gold nanoparticles for the perfectly matched DNA, which enabled a detection of perfectly matched DNA from the single point-mutated one. Our results indicate that a change in the electrostatic interaction is responsible for the selective aggregation of gold nanoparticles upon the addition of DNA. This suggest...

Study of polydimethylacrylamide- and polydiethylacrylamide-adsorbed coatings on fused silica capillaries and their application in genetic analysis

The adsorption of two polymers (polydimethylacrylamide and polydiethylacrylamide) on the inner surface of a fused silica of capillary (or wafer) was investigated by means of atomic force microscopy (AFM), multi-angle laser light scattering (MALLS) technique, and by measuring the electroosmotic flow (EOF) and contact angle. The AFM images showed that PDMA and PDEA tightly adsorbed on the fused silica surface and formed stable coatings. The contact angle data demonstrated that the polymer-adsorbed coatings have different hydrophobicities, which are related to the structures of the polymers. The adsorbing capability and stability of the adsorption coating, perhaps, were mainly dependent on the hydrogen bond force between oxygen atom on the carbonyl group of polymers and the hydroxyl group of the silica surface, and the hydrophobic nature of polymers. Our data also illustrated that the polymer-adsorbed coatings efficiently suppressed the EOF and the adsorption of DNA fragments on the capillary surface. These polymers were successfully used as sieving media in capillary electrophoresis of DNA fragments and detection of single point mutation of C677T from human methylenetetrahydrofolate reductase (MTHFR) gene.

Detection of Apolipoprotein B mRNA Editing by Peptide Nucleic Acid Mediated PCR Clamping

Apolipoprotein B (apoB) mRNA editing leads to a single base change in its mRNA and the production of apoB-48. Currently, the degree of apoB mRNA editing is analyzed by the RT-PCR primer extension method. While this method is quantitative, it is labor intensive, utilizes radioactivity for labeling and may not be sensitive enough to discriminate between low levels of editing and inherent assay background levels. Peptide nucleic acid (PNA) oligonucletides have been used in single point mutation detection through PCR clamping. In the present work, we developed a PCR based assay which can detect the single base change responsible for the apoB-48 production. We found that as low as 0.5% of the edited form can be clearly detected by PNA mediated PCR clamping. When combined with the primer extension assay, an approximately 180-fold enrichment of the edited percentage is observed, reflecting selected PCR amplification of templates containing the edited base.

Pna Array Technology in Molecular Diagnostics

A comparative study using immobilised DNA and PNA oligomers demonstrates the suitability of PNA molecules as sequence specific capture probes in the detection of single point mutations in a DNA analyte and in the analysis of complex analyte mixtures.

Competitive assay to improve the specificity of detection of single-point mutations in alpha 1-antitrypsin deficiency

Allele-specific oligonucleotides are used widely for the detection of single point mutations in genes. A modification of this assay based on competition has been developed for detection of the Z mutation of alpha 1-antitrypsin (alpha 1-AT). The normal alpha 1-AT allele is referred to as M, and the Z mutation arises from a single base substitution. Amplified DNA products corresponding to homozygous M, heterozygous MZ, and homozygous Z obtained by the polymerase chain reaction were incubated with a twofold molar excess of unlabeled oligonucleotide prior to hybridization with a radiolabeled oligonucleotide. Thus, initial incubation with unlabeled M-specific oligonucleotide was followed by hybridization with radiolabeled Z-specific oligonucleotide, and vice versa. This assay increased the specificity of single-point mutation detection three- to four-fold. Furthermore, specific hybridization was obtained at a lower temperature as a consequence of improving the signal-to-noise ratio.