Length Of DNA Molecules Research Articles

Artificial Intelligence-Enhanced Analysis of Genomic DNA Visualized with Nanoparticle-Tagged Peptides under Electron Microscopy.

DNA visualization has advanced across multiple microscopy platforms, albeit with limited progress in the identification of novel staining agents for electron microscopy (EM), notwithstanding its ability to furnish a broad magnification range and high-resolution details for observing DNA molecules. Herein, a non-toxic, universal, and simple method is proposed that uses gold nanoparticle-tagged peptides to stain all types of naturally occurring DNA molecules, enabling their visualization under EM. This method enhances the current DNA visualization capabilities, allowing for sequence-specific, genomic-scale, and multi-conformational visualization. Importantly, an artificial intelligence (AI)-enabled pipeline for identifying DNA molecules imaged under EM is presented, followed by classification based on their size, shape, or conformation, and finally, extraction of their significant dimensional features, which to the best of authors' knowledge, has not been reported yet. This pipeline strongly improved the accuracy of obtaining crucial information such as the number and mean length of DNA molecules in a given EM image for linear DNA (salmon sperm DNA) and the circumferential length and diameter for circular DNA (M13 phage DNA), owing to its image segmentation capability. Furthermore, it remained robust to several variations in the raw EM images arising from handling during the DNA staining stage.

Controllable concentric electric field line distribution for simultaneous separation of DNA

An approach for the controllable separation and concentration of nucleic acid using a circular nonuniform electric field was proposed and developed. Using six different lengths of DNA molecules as standard samples, the distribution of the gradient electric field was increased from the outer circular electrode to the inner rod-shaped electrode, contributing to the migration of DNA molecules at a velocity gradient towards the region with the strongest inner electric field. The DNA molecules were arranged in a distribution of concentric circles that aligned with the distribution of concentric equipotential lines. The concentration of DNA multiplied with the alternation of radius. As a result, this platform allowed simultaneous DNA separation, achieving a resolution range of 1.17–3.03 through an extended electrophoresis time, resulting in enhanced concentration factors of 1.08–6.27. Moreover, the manipulation of the relative height of the inner and outer electrodes enabled precise control over the distribution and the deflection degree of electric field lines, leading to accurate control over DNA deflection.

Влияние длины молекул и природы среды на фрагментацию ДНК ультразвуком

Fragmentation of DNA by ultrasound is the most optimal method of artificial degradation of DNA molecules. It requires no reagents and makes it possible to obtain "pure" DNA samples for further use. However, despite an active usage of ultrasonic fragmentation in studies, a number of issues related to the applicability of mechanically degraded DNA for standard molecular biological operations remain poorly studied. This report presents data on the effect of the size of DNA molecules (on DNA amplicons) and the composition of aqueous solutions on the efficiency of DNA destruction by ultrasound. It has been shown that the rate of DNA fragmentation positively correlates with an increase in the length of molecules. In solutions with high ionic strength, the rate of fragmentation increases dramatically. The presence of organic solvents that do not affect hybridization slightly reduces the rate of ultrasonic destruction. With an increase in the length of DNA molecules, the influence of denaturing agents changes and leads to an increase in the rate of fragmentation. dsDNA molecules smaller than one persistent length (about 150 bp) are not fragmented.

Linked‐read sequencing enables haplotype‐resolved resequencing at population scale

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences - including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps - are still limited by the lack of high-quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype-resolved genome resequencing at population scale, we investigated properties of linked-read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high-coverage data (46-68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25-4.6Mb (N50) and 0.27-0.72Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7Mb/1Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher-quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase-sized genomes like birds, linked-read sequencing at moderate depth opens an affordable avenue towards haplotype-resolved genome resequencing at population scale.

Atomic force microscopy study of EDTA induced desorption of metal ions immobilized DNA from mica surface

Adsorption of DNA molecules onto substrate, such as mica, is well documented. However, desorption of the immobilized DNA molecules from substrate, which is also an important aspect of DNA behavior on substrate, has not been paid much attention. Here, DNA molecules were first immobilized on mica surface by using divalent metal ions as the bridge agents. Desorption of the immobilized DNA from mica surface was realized via ethylenediamine tetraacetic acid disodium salt (EDTA) treatment. EDTA is a chelating agent; it can remove the bridging metal ions between DNA and mica, which leads to the release of DNA molecules from mica substrate. The divalent metal ions assisted DNA adsorption onto mica surface and the EDTA induced DNA desorption from mica surface were followed by atomic force microscopy (AFM). Randomly dispersed DNA strands and DNA networks are two distinct adsorption morphologies of DNA on mica surface and their desorption processes from mica surface induced by EDTA are also different. Other factors that influence the EDTA-induced DNA desorption, such as type of bridging metal ion and DNA molecule length, have also been systematically studied. Moreover, EDTA treatment has no effect on the integrity of DNA molecule. The EDTA induced desorption of metal ions immobilized DNA from mica surface is simple and effective, which has potential applications in DNA separation and purification, DNA biophysics, and DNA-based nanotechnology.

Kinetics of double strand breaks of DNA in tritiated water evaluated using single molecule observation method

Double strand breaks (DSBs) of DNA molecules in tritiated water was examined under sterilized and non-sterilized conditions using a single molecule observation method. The genome DNA of bacteriophage T4 GT7 was immersed in sterilized tritiated water (5.2 MBq/cm3) and non-sterilized tritiated water (4.2 MBq/cm3) for 1, 7 and 14 day(s). Then the length of DNA molecules was measured using a fluorescence microscope after intercalation of fluorescent dye. The dose rate was 1.4–1.7 × 10−2 Gy/h and the dose level was 0.41–5.8 Gy. The rate of DSBs induced by β-rays from tritium was successfully evaluated under the sterilized conditions and the value comparable with the DSB rate under γ-ray irradiation (Noda et al., Scientific Reports 7 (2017) 8557) was obtained. The length of DNA molecules in non-sterilized tritiated water was clearly shorter than that in the sterilized tritiated water. This observation suggested that the effects of tritium was far weaker than that of microorganisms (e.g. bacteria) and impurities in water even at the tritium concentration as high as 5.2 MBq/cm3.

A sensitive biosensor based on optical bistability in a semiconductor quantum dot-DNA nanohybrid

We propose a scheme of a sensitive biosensor based on optical bistability (OB) in a biological semiconductor quantum dot (SQD)-DNA coupled nanohybrid. By plotting bistability phase diagrams within the system’s parameter space, we show that the bistable effect can be switched on or off by only tuning the pumping intensity or the detuning of the pump field from the exciton resonance. Simultaneously, we reveal that the bistability only exists in the strong SQD-DNA coupling regime. Specifically, we derive the matching relation between the excitation conditions and the coupling strength when the bistability just appears. Moreover, we demonstrate that the OB is highly sensitive to the SQD-DNA coupling strength which is strongly correlated to the length of DNA molecules and DNA concentration. This investigation may be useful for opening a new way to build optical switches and providing a novel and convenient platform to detect DNA molecules.

Acousto-optical tweezers for stretch of DNA molecule

The appearance of microlenses in the amorphous crystal Ge33As12Se33 modulated by the acoustic waves with intensity of 107 W/m2 and frequency of (200–400) MHz is presented. The flexibility of microlens, i.e. the dependence of its focal point in 3D space on the parameters of acoustic wave is studied. Basing on flexibility of microlens, a model of acousto-optical tweezers to stretch the λ-phage WLC DNA molecule is proposed. And then the control process of stretched length of DNA molecule in 3D space of the water (fluid) by calibration of the acoustic frequency is numerically observed and discussed. The obtained results show the possibility to use the acousto-optical tweezers to control the stretched length of WLC DNA molecule with high fineness.

Next generation mapping reveals novel large genomic rearrangements in prostate cancer.

Complex genomic rearrangements are common molecular events driving prostate carcinogenesis. Clinical significance, however, has yet to be fully elucidated. Detecting the full range and subtypes of large structural variants (SVs), greater than one kilobase in length, is challenging using clinically feasible next generation sequencing (NGS) technologies. Next generation mapping (NGM) is a new technology that allows for the interrogation of megabase length DNA molecules outside the detection range of single-base resolution NGS. In this study, we sought to determine the feasibility of using the Irys (Bionano Genomics Inc.) nanochannel NGM technology to generate whole genome maps of a primary prostate tumor and matched blood from a Gleason score 7 (4 + 3), ETS-fusion negative prostate cancer patient. With an effective mapped coverage of 35X and sequence coverage of 60X, and an estimated 43% tumor purity, we identified 85 large somatic structural rearrangements and 6,172 smaller somatic variants, respectively. The vast majority of the large SVs (89%), of which 73% are insertions, were not detectable ab initio using high-coverage short-read NGS. However, guided manual inspection of single NGS reads and de novo assembled scaffolds of NGM-derived candidate regions allowed for confirmation of 94% of these large SVs, with over a third impacting genes with oncogenic potential. From this single-patient study, the first cancer study to integrate NGS and NGM data, we hypothesise that there exists a novel spectrum of large genomic rearrangements in prostate cancer, that these large genomic rearrangements are likely early events in tumorigenesis, and they have potential to enhance taxonomy.

Owl pellets: a wise DNA source for small mammal genetics

AbstractPellets of raptors are an important source for the study of the taxonomy, phylogeography and ecological diversity of small vertebrates. Since birds of prey are efficient collecting agents for both rare species and those reluctant to enter traps, they offer an important complement to traditional trapping efforts in the field. The possibility of using bones and teeth recovered from pellets as a convenient alternative DNA source for genetic studies requires a more complete understanding of the DNA preservation of these partially digested remains. It is not yet clear, for example, to which degree DNA is preserved and how heterogeneous DNA preservation is in the different skeletal remains found in pellets since systematic quantitative analyses are missing. Here, we attempt to improve this knowledge by quantifying the degradation of DNA during the digestion process of raptors using 62 individual rodent bones and teeth from different pellets. We show a very high variability in DNA preservation between bones. Interestingly, variability between bones within the same pellet is much higher than that found between pellets, which has major consequences for genetic studies as it implies that bones from the same pellet that could belong to different individuals/species should be tested individually when multiple markers are analyzed. We find that neither the identity of the skeletal part nor microscopic observation of the degree of digestion are useful proxies for DNA preservation, and we thus recommend the use of screening strategies to identify the most suitable bones to include in analyses. We conclude that pellets of owls, whether freshly collected or from natural history collections, can be a practical noninvasive source for genetic studies of small vertebrates provided the design of the study takes into account the short DNA molecule length, the low quantity of DNA preserved and the heterogeneity in DNA preservation.

Assessing the performance of the Oxford Nanopore Technologies MinION

The Oxford Nanopore Technologies (ONT) MinION is a new sequencing technology that potentially offers read lengths of tens of kilobases (kb) limited only by the length of DNA molecules presented to it. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. It has numerous prospective applications including improving genome sequence assemblies and resolution of repeat-rich regions. Before such a technology is widely adopted, it is important to assess its performance and limitations in respect of throughput and accuracy. In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G+C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2kb and 1kb respectively, while the longest single read was 98kb. The whole length of a 5kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of de novo assembly when used in conjunction with Illumina MiSeq data.

Biophysical studies of the effect of high power ultrasound on the DNA solution

Stability and molecular size of the DNA double helical structure were studied on an aqueous solution of DNA after exposure to high power doses of continuous wave ultrasound at frequency of 20 kHz. Thermal transition spectrophotometry (UV-melting), constant-field gel electrophoresis (CFGE), differential scanning calorimetry (DSC) and dielectric properties measurements were used to evaluate the ultrasound-induced changes in the DNA double helical structure. The thermal transition spectrophotometry (UV-melting) and differential scanning calorimetry (DSC) results showed that ultrasound power caused loss of DNA double helical structure and the DNA double strands melting temperature decreased as the ultrasound power increased, indicating a decrease in the stability of the double helical structure of DNA. The constant-field gel electrophoresis (CFGE) results showed that the molecular size of the DNA fragments decreased as the ultrasound power increased. The dielectric data in the frequency range from 20 Hz to 100 kHz for the native DNA showed that dispersion at frequency of about 500 Hz resulted from polarization induced by counterions. The decrease in the dielectric increment indicated a decrease in length of DNA molecule after exposure to ultrasound power.

On'mechanism'of'inactivation'of'bio-particles'by'the'plasma'exposure'and'evaluation'of'the'toxicity'using'single'DNA'molecules

NTP can also inactivate viruses. In the previous research, critical damages for the inactivation of λ phage were done on coat proteins and M13 phage on DNA. These results were obtained with wet phage sample [1]. We also tried to use dry sample. We used bacteriophage φX174 which is resistant to drying. When the bacteriophage is inactivated by NTP, the damage should exist either on viral nucleic acids or coat proteins or both. It is possible to extract the nucleic acids from the bacteriophage so that the damage to the DNA can be separately analyzed to exclude the effect of the proteins. The DNA extracted from the plasma-exposed phage can be assayed its plaque forming activity by transfection. The coat proteins were analysed by SDS-PAGE. Using these assays, the damages of viral DNA and coat proteins have been determined separately. The results for the dry φX174 phage inactivated by the exposure to DBD showed both DNA and coat proteins were damaged and the critical damage were done to the coat proteins. This is similar to the result of the wet sample. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water. References

Obtaining Quantitative Parameters of DNA-Ligand Cooperative Binding from Persistence Length Measurements

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We have measured the persistence length of DNA molecule for cationic and neutral beta-cyclodextrin binding, using optical tweezers. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of total ligand concentration, which fits very well our data of the DNA-cationic beta-cyclodextrin and the DNA-HU protein data available in the literature. The fit returns the values of the local persistence lengths, the dissociation constant and the degree of cooperativity for both sets of data. In both cases the persistence length behaves non-monotonically as a function of total ligand concentration. We discuss some physical mechanisms for these binding processes and their interplay with DNA flexibility.ReferencesM. S. Rocha et al., J. Chem. Phys. 127 (10), 105108 (2007);J. van Noort et al., PNAS 101 (18), 6969 (2004);D. Sagi et al., J. Mol. Biol., 341, 419 (2004).Sponsors: CNPq, Fapemig, Capes, Pronex-Facepe and INCT-FCx

Detection and Damage-analysis of Bio-Particles and for Safety-Evaluation of Plasma-treated water using DNA-manipulation

ABSTRACTEffect of non-thermal plasma (NTP) on bio-particles has been studied using Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and bacteriophages. NTP has been used, and states of different biological components were monitored during the course of the exposure. Analysis of green fluorescent protein (GFP), introduced into E.coli cells proved that NTP causes a prominent protein damages without cutting peptide bonds. We have developed a biological assay which evaluates in vivo DNA damage of the bacteriophages. Different doses of the plasma were applied to wet state of λ phages. From the plasma-exposed λ phages, DNA was purified and subjected to in vitro DNA packaging reactions. The re-packaged phages consist of the DNA from discharged phages and brand-new coat proteins. Survival curves of the re-packaged phages showed extremely large D value (D = 25 s) compared to the previous D value (D = 3 s) from the discharged phages. The results indicate that DNA damage hardly contributed to the inactivation, and the damage in coat proteins is responsible for inactivation of the phages. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasmainduced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water.

Single-molecule measurement of strand breaks on large DNA induced by atmospheric pressure plasma jet

We report a single-molecule-based analysis of strand breakages on large DNA molecules induced by an atmospheric pressure plasma jet. We exposed DNA solution to an argon plasma jet; single-molecule observation that involved molecular combing was then used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. For strand breaks on large DNA molecules the rate was estimated.

Conformational analysis and estimation of the persistence length of DNA using atomic force microscopy in solution

The worm-like chain model describes the mechanical properties of semi-flexible polymers by introducing a certain correlation length along the contour. This correlation length is called the persistence length. Using atomic force microscopy in solution, we performed measurements of the persistence length of DNA molecules. We found good agreement between the theoretical model and experimental data. However, the measured persistence length values in solution differ from those found by several authors using the same technique but in dry air. In order to determine the contribution of the electrostatic persistence length to the total persistence length, we varied the salt concentration. We found a large discrepancy between the Odijk, Skolnick and Fixman theory and our measurements. The effect of divalent ions and the omission in the theory of the dependence of non-electrostatic persistence length on salt concentration are qualitatively invoked.

Analysis of DNA coverage using enzymatic cleavage of fluorescent labels

In this study, a method to determine the degree of DNA coverage on gold surface using a fluorescent dye and Deoxyribonuclease I (DNase I) was developed. Gold surfaces were first functionalized with fluorescent dye labeled DNA, which was followed by DNase I digestion. The number of released fluorescent dyes represented the quantity of DNA immobilized. The use of DNase I to enzymatically cleave the fluorescent dye decreased the time needed for quantification to 1 h. In this way, the effect of a back-filler, DNA concentration, incubation time and spacer on DNA immobilization via gold-thiol chemistry was examined. The concentration of oligonucleotides and the ionic environment were the major factors determining DNA density. We also found that the length of DNA molecule influenced the rate of immobilization on Au. In addition, mixed self-assembled monolayer (mixed-SAM) with various composition and the the effect on DNA density was investigated.

A kinetic analysis of strand breaks on large DNA induced by cigarette smoke extract

We report a kinetic analysis of strand breakages on large DNA molecules induced by cigarette smoke extract (CSE), an extract of soluble cigarette smoke components. Previously, this DNA damage was analyzed by agarose gel electrophoresis, whereas we used fluorescence to kinetically analyze damage to individual DNA molecules. CSE caused a marked change in length of DNA molecules. The rate of CSE-induced double-strand breakage on large random-coiled DNA molecules was determined using a simple theoretical model, allowing the facile estimation of the rate of double-strand breaks on large DNA molecules.

DNA Dynamics in Nanoscale Confinement under Asymmetric Pulsed Field Electrophoresis

Unbending: Asymmetric pulsed field electrophoresis of DNA was conducted in a nanoporous array formed by colloidal self-assembly of nanoparticles inside a microfluidic system. In confinement smaller than the persistence length of DNA molecules, DNA is maintained in highly stretched conformation. A model correlating pulse frequency, electric field, and molecular size with the observed angular dispersion was developed and experimentally verified.