Sequencing Of DNA Molecules Research Articles

A biochemistry platform to incorporate replication forks, DNA lesions, and nucleosome arrays to single-molecule experiments.

Fundamental biological processes such as DNA replication, DNA repair, and chromatin organization are precisely regulated in space and time by sophisticated multiprotein molecular machinery. The high spatiotemporal resolution of optical tweezers combined with single molecule fluorescence microscopy enables real-time visualization of these processes while simultaneously measuring their enzymatic activity via detection of mechanical manipulation of the DNA substrates. Having a dedicated and relevant DNA substrate is critical to determining the relevant molecular mechanisms in a single-molecule experiment. Here, we will share our biochemical solutions for various applications on double-stranded and single-stranded DNA. We also present our newly developed biochemical tools to incorporate replication forks, DNA lesions, and specific sequences in DNA molecules tethered between two beads. Our next developments aim to combine multiple DNA features in a single DNA molecule, such as a replication fork in the presence of DNA lesions or in the context of a nucleosome array. These advances will allow more complex experimental set ups and increase the capability to mimic cellular biological processes at a single molecule level.

ПЕРСОНАЛИЗИРОВАННАЯ ТЕРАПИЯ РАКА – ДОСТИЖЕНИЯ ГЕНЕТИЧЕСКОЙ МЕДИЦИНЫ

At present, cancer is a rising dreadful issue all around the world, as it is among one of the main leading causes for mortality globally. Recent improvements in genetic engineering called genome sequencing provides a different ap- proach in the treatment of cancer. Genome sequencing is a technique used for determining the complete composition of the DNA sequence of a genome. The cancer genomic project and the cancer genome atlas are initiatives taken by different countries to analyze and store the genomic sequence variations of different tumor cells from a diverse popula- tion. Sequencing malignant cells have pointed the unique abnormalities like mutations in the genes, which shows the exact cause for the uncontrolled cell division in many types of malignancies. We discuss the recent effective method called next-generation sequencing which has the ability to sequence DNA molecules in thousands, simultaneously, rapidly, and cost- effectively. NGS can detect all types of mutations and chromosomal abnormalities. This information provides a detailed and improved understanding of different types and subtypes of cancer from their molecular basis. The process includes sequencing of both tumor and normal genome from the same individual, and then are examined and verified. This data is used to provide a personalized treatment routine for that specific patient which can improve their prognosis. The various challenges and limitations of NGS which are hindering the advancement and the prob- ability of using it in clinical oncology are also discussed which include ethical problems and abundant data generated causing an issue in data analysis. We outline the recent promising developments in implementing genomic science in the personal and targeted treatment of cancer for better prognosis and plausible cure for malignancy in the near future.

Non-Euclidean Biosymmetries and Algebraic Harmony in Genomes of Higher and Lower Organisms

The article is devoted to the study of the relationship of non-Euclidean symmetries in inherited biostructures with algebraic features of information nucleotide sequences in DNA molecules in the genomes of eukaryotes and prokaryotes. These genomic sequences obey the universal hyperbolic rules of the oligomer cooperative organization, which are associated with the harmonic progression 1/1, 1/2, 1/3,.., 1/n. The progression has long been known and studied in various branches of mathematics and physics. Now it has manifested itself in genetic informatics. The performed analysis of the harmonic progression revealed its connection with the cross-ratio, which is the main invariant of projective geometry. This connection consists in the fact that the magnitude of the cross-ratio is the same and is equal to 4/3 for any four adjacent members of this progression. The long DNA nucleotide sequences have fractal-like structure with so called epi-chains, whose structures are also related to the harmonic progression and the projective-geometrical symmetries. The received results are related additionally to a consideration of DNA double helix as helical antenna. This fact of the connection of genetic informatics with the main invariant of projective geometry can be used to explain the implementation of some non-Euclidean symmetries in genetically inherited structures of living bodies.

Harmonic Progression in Bioinformatics and Recurrent Series in Inherited Biostructures

The article is devoted to the study of new approaches to the development of mathematical models in genetic biomechanics, which studies the structural relationships of the genetic coding system with genetically inherited biological forms. More specifically, we are talking about models based on the recurrent harmonic progression whose connection with the information sequences of DNA molecules in the genomes of higher and lower organisms was recently revealed. In particular, the article describes previously unknown connections of the function of natural logarithms with the structures of the molecular genetic system, which allow modelling the main psychophysical logarithmic law by Weber-Fechner and also many other logarithmic structures in genetically inherited biological systems. In physics, the harmonic progression is traditionally considered, first of all, as related to standing waves in resonators. Our results are correlated with Frohlich’s vibration-resonant theory about collective quantum effects and long-range communication in biological systems.

DNA of Things: how a plastic bunny got DNA

The ever-increasing amount of digital data has led scientists to look for new ways of storing information efficiently. In the last years, a new field of research has evolved around storing information in the sequence of DNA molecules. We have shown a new approach, which allows us to encode data in DNA and store it in everyday objects like coffee cups, reading glasses or 3D-printed bunnies. - submission by Julian Koch, Robert N. Grass

DNA sequencing by nanopores: achievements and prospects

After years of development, the use of nanopore as a sensor to sequence DNA molecules is now a viable and promising possibility. Single base pair detection during DNA transport enables to record ultra-long threads with high parallelization and rates. I will present in this review the current methodologies based on electrical detection and biological nanopores and the new methods based on solid state nanopores and optical detection.

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome.

Advances in long-read single molecule sequencing have opened new possibilities for ‘benchtop’ whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two Caenorhabditis elegans strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type C. elegans genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the C. elegans reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.

BIOPHYSICAL BASES OF GENOME ORGANIZATION

Aim - the analysis of nucleotide sequences of DNA molecules and the bases of the information storage with the help of DNA. Material and methods - the study is based on Markov chain theory and Bayesian Information Criterion. Results. Principles of genetic code construction were investigated. Specific nucleotide sequences were analyzed using Markov chain theory; the method of sequencing nucleotide sequences was described. Conclusion. A nucleotide sequence has certain restrictions associated with complementarity of the bases along DNA chain. These restrictions at the level of triplet sequence can be eliminated by degeneracy of the genetic code.

Measurements of single nucleotide electronic states as nanoelectronic fingerprints for identification of DNA nucleobases, their protonated and unprotonated states, isomers, and tautomers.

Several nanoelectronic techniques have been explored to distinguish the sequence of nucleic acids in DNA macromolecules. Identification of unique electronic signatures using nanopore conductance, tunneling spectroscopy, or other nanoelectronic techniques depends on electronic states of the DNA nucleotides. While several experimental and computational studies have focused on interaction of nucleobases with different substrates, the effect of nucleic acid biochemistry on its electronic properties has been largely unexplored. Here, we present correlated measurements of frontier molecular orbitals and higher-order electronic states for four DNA nucleobases (adenine, cytosine, thymine, and guanine), and first-principle quantum chemical density functional theoretical (DFT) computations. Using different pH conditions in our experiments, we show that small changes in the biochemical state of these nucleic acids strongly affect the intrinsic electronic structure, measured using scanning tunneling spectroscopy (STS). In our experimental measurements and computations, significant differences were observed between the position of frontier orbitals and higher-energy states between protonated and unprotonated nucleic acids, isomers, and different keto-enol tautomer's formed in these nucleotides, leading to their facile identification. Furthermore, we show unique "electronic fingerprints" for all nucleotides (A, G, T, C) using STS, with most distinct states identified at acidic pH. These results can have important implications for identification of nucleic acid sequences in DNA molecules using a high-throughput nanoelectronic identification technique.

Simultaneous extraction of stratigraphic sequences using iterative seismic DNA detection

We developed a new stratigraphic interpretation methodology enabling extraction of stratigraphic sequences of surfaces. This methodology consisted of two main components. The first component was inspired by DNA search technology used in bioinformatics in which the goal was to detect base-pair sequences in DNA molecules. The base-pair sequences may be modified by mutations, so the search technology must be able to take such mutations into account. We have adapted this technology to search for seismic reflection sequences, acknowledging that seismic reflections were likely to vary laterally that were somewhat analogs to mutations in bioinformatics. One example of a seismic reflection sequence was a geologically sorted sequence of seismic reflectors. The second component of the workflow was how to connect the DNA hits into continuous surfaces. A quality metric was used to steer the connection process of the DNA hits in a sorted order. This workflow enabled the user to extract a sequence of surfaces simultaneously, which is crucial in seismic stratigraphic interpretation.

Visible periodicity of strong nucleosome DNA sequences

Fifteen years ago, Lowary and Widom assembled nucleosomes on synthetic random sequence DNA molecules, selected the strongest nucleosomes and discovered that the TA dinucleotides in these strong nucleosome sequences often appear at 10–11 bases from one another or at distances which are multiples of this period. We repeated this experiment computationally, on large ensembles of natural genomic sequences, by selecting the strongest nucleosomes – i.e. those with such distances between like-named dinucleotides, multiples of 10.4 bases, the structural and sequence period of nucleosome DNA. The analysis confirmed the periodicity of TA dinucleotides in the strong nucleosomes, and revealed as well other periodic sequence elements, notably classical AA and TT dinucleotides. The matrices of DNA bendability and their simple linear forms – nucleosome positioning motifs – are calculated from the strong nucleosome DNA sequences. The motifs are in full accord with nucleosome positioning sequences derived earlier, thus confirming that the new technique, indeed, detects strong nucleosomes. Species- and isochore-specific variations of the matrices and of the positioning motifs are demonstrated. The strong nucleosome DNA sequences manifest the highest hitherto nucleosome positioning sequence signals, showing the dinucleotide periodicities in directly observable rather than in hidden form.

Detecting the translocation of DNA through a nanopore using graphene nanoribbons

Solid-state nanopores can act as single-molecule sensors and could potentially be used to rapidly sequence DNA molecules. However, nanopores are typically fabricated in insulating membranes that are as thick as 15 bases, which makes it difficult for the devices to read individual bases. Graphene is only 0.335 nm thick (equivalent to the spacing between two bases in a DNA chain) and could therefore provide a suitable membrane for sequencing applications. Here, we show that a solid-state nanopore can be integrated with a graphene nanoribbon transistor to create a sensor for DNA translocation. As DNA molecules move through the pore, the device can simultaneously measure drops in ionic current and changes in local voltage in the transistor, which can both be used to detect the molecules. We examine the correlation between these two signals and use the ionic current measurements as a real-time control of the graphene-based sensing device.

Individual RNA Base Recognition in Immobilized Oligonucleotides Using a Protein Nanopore

Protein nanopores are under investigation as key components of rapid, low-cost platforms to sequence DNA molecules. Previously, it has been shown that the α-hemolysin (αHL) nanopore contains three recognition sites, capable of discriminating between individual DNA bases when oligonucleotides are immobilized within the nanopore. However, the direct sequencing of RNA is also of critical importance. Here, we achieve sharply defined current distributions that enable clear discrimination of the four nucleobases, guanine, cytosine, adenine, and uracil, in RNA. Further, the modified bases, inosine, N(6)-methyladenosine, and N(5)-methylcytosine, can be distinguished.

Selection of a reference sequence for the determination of nucleotide sequences in DNA molecules

A fragment of the DNA sequence of the pUC18 plasmid is proposed as a reference sequence for the calibration and metrological assurance of genome analyzers. This fragment consists of 271 nucleotide pairs and is obtained by a reference method. This sequence is optimal for determining the metrological characteristics of genome analyzers in light of its stability and the existence of several segments with repeating nucleotides. An example is given for the calculation of a genome analyzer characteristic, namely, the probability of error in measurement of the genome sequence.

A Stacked Graphene-Al2O3 Nanopore Architecture for DNA Detection

Nanopore based DNA sensing methods use electrophoresis to drive negatively charged molecules through nanometer sized pores and monitor the change in ionic current to examine the length or sequence of DNA molecules. It is an inexpensive and attractive alternative to traditional sequencing and analysis technologies as it is a label-free, amplification-free, single-molecule approach that can be scaled for high-throughput DNA analysis. Nanopores have been shown to be a versatile platform capable of sensing subtle changes in the structure of biomolecules.We report the development of robust multilayered graphene-Al2O3 nanopore platform for sensitive detection of DNA and DNA-protein complexes. Graphene-Al2O3 nanolaminate membranes are formed by sequentially depositing layers of graphene and Al2O3 via atomic layer deposition. Subsequently, nanopores were formed in these stacked membranes using an electron-beam sculpting process. The resulting nanopore architecture is mechanically robust and exhibits state-of-the-art low electrical noise. The platform was used for detection of both DNA and protein DNA complexes and showed the ability to differentiate structural changes like folding and protein binding. The structure can also be used for electrostatic potential control at the nanopore surface to guide ionic current and possibly translocation velocity. The combination of simplicity and versatility makes nanopores an attractive platform for medical diagnostic applications and DNA sequencing.

Dynamic cluster-scaling in DNA

It is shown that the nucleotide sequences in DNA molecules have cluster-scaling properties (discovered for the first time in turbulent processes [K.R. Sreenivasan, A. Bershadskii, J. Stat. Phys. 125 (2006) 1141]. These properties are relevant to both types of nucleotide pair-bases interactions: hydrogen bonds and stacking interactions. It is shown that taking into account the cluster-scaling properties can help to improve heterogeneous models of the DNA dynamics. Two human genes: BRCA2 and NRXN1, have been considered as examples.

Nanomechanical behaviors of microcantilever-based single-stranded DNA chips induced by counterion osmotic effects

Experiments show that deflections of microcantilever-DNA chip can be induced by many factors, such as grafting density, hybridization efficiency, concentration, length and sequence of DNA molecules, buffer salt concentration, time, and temperature variation. However, there are few theoretical works on microcantilever-DNA chips. The present paper is aimed to study the influence of counterion effects of single-stranded DNA (ssDNA) polyelectrolyte solution on the nanomechanical behaviors of microcantilever-based ssDNA chips during packing process. First, the effect of osmotic pressure induced by ingress of counterions into DNA brush structures is studied with Hagan's model for a cylindrical polyelectrolyte brush system on the basis of Poisson-Boltzmann distribution hypothesis. Second, Zhang's two-variable method for a laminated cantilever is used to formulate a four-layer energy model for ssDNA chips with weak interactions. Third, the influence of grafting density, ssDNA chain length, and salt concentration on packing deflection is investigated using the principle of minimum energy. The predictive tendency is qualitatively similar to those observed in some related ssDNA chip experiments. The difference between the four-layer model and the simplified two-layer model for ssDNA chips is also discussed.

Long-range correlation and charge transfer efficiency in substitutional sequences of DNA molecules

We address the relation between long-range correlations and coherent charge transfer in substitutional DNA sequences using the transfer matrix approach. The substitutional sequences exhibit long-range correlations and show good transmittivity in comparison with uncorrelated random ones. It is found that the charge transfer efficiency varies for different substitutional sequences and many will present electronic delocalization in the system. Further, the resistivity for substitutional sequences may range from decreasing with the length, length independence, or increasing with the length. The conduction mechanisms of various behaviors observed for these sequences are analyzed.

Detection of cruciform extrusion in DNA by temperature-gradient gel electrophoresis

Repetitive sequences in DNA molecules, some of which are palindromic, tend to form stable cruciforms. These are frequently located in promoter regions of a specific operon and origin of replication. Temperature gradient gel electrophoresis can be used to distinguish among various supercoiled DNA topoisomers and to ascertain whether or not the cruciform motif has been extruded. In the current study, this technique is implemented for the first time to address the role of temperature in cruciform extrusion from plasmids.

In vitro selection of molecular beacons.

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.