Free DNAs Research Articles

Immune markers of severe acute pancreatitis.

Acute pancreatitis is a common acute inflammatory disorder of the pancreas, and its incidence has been increasing worldwide. Approximately 10% of acute pancreatitis progresses to severe acute pancreatitis (SAP), which carries significant morbidity and mortality. Disordered immune response to pancreatic injury is regarded as a key event that mediates systemic injury in SAP. In this article, we review recent developments in immune biomarkers of SAP and future directions for research. Given the importance of the NLRP3-inflammasome pathway in mediating systemic inflammatory response syndrome and systemic injury, recent studies have investigated associations of SAP with systemic levels of activators of NLRP3, such as the damage associated molecular patterns (DAMPs) for the first time in human SAP. For example, circulating levels of histones, mitochondrial DNAs, and cell free DNAs have been associated with SAP. A panel of mechanistically relevant immune markers (e.g., panel of Angiopoeitin-2, hepatocyte growth factor, interleukin-8 (IL-8), resistin and sTNF-α R1) carried higher predictive accuracies than existing clinical scores and individual immune markers. Of the cytokines with established relevance to SAP pathogenesis, phase 2 trials of immunotherapies, including tumor necrosis factor (TNF)-alpha inhibition and stimulation of IL-10 production, are underway to determine if altering the immunologic response can reduce the severity of acute pancreatitis (AP). Circulating systemic levels of various DAMPs and a panel of immune markers that possibly reflect activities of different pathways that drive SAP appear promising as predictive biomarkers for SAP. But larger multicenter studies are needed for external validation. Studies investigating immune cellular pathways driving SAP using immunophenotyping techniques are scarce. Interdisciplinary efforts are also needed to bring some of the promising biomarkers to the bedside for validation and testing for clinical utility. Studies investigating the role of and characterization of altered gut-lymph and gut-microbiota in severe AP are needed.

Transient interactions modulate the affinity of NF-κB transcription factors for DNA

The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 μs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA.

Isomerization Pathways of a Mismatched Base Pair of A:8OG in Free Duplex DNA.

The A:8OG base pair (bp) is the outcome of DNA replication of the mismatched C:8OG bp. A high A:8OG bp population increases the C/G to A/T transversion mutation, which is responsible for various diseases. MutY is an important enzyme in the error-proof cycle and reverts A:8OG to C:8OG bp by cleaving adenine from the A:8OG bp. Several X-ray crystallography studies have determined the structure of MutY during the lesion scanning and lesion recognition stages. Interestingly, glycosidic bond (χ) angles of A:8OG bp in those two lesion recognition structures were found to differ, which implies that χ-torsion isomerization should occur during the lesion recognition process. In this study, as a first step to understanding this isomerization process, we characterized the intrinsic dynamic features of A:8OG in free DNAs by a free energy landscape simulation at the all-atom level. In this study, four isomerization states were assigned in the order of abundance: Aanti:8OGsyn > Aanti:8OGanti > Asyn:8OGanti ≈ Asyn:8OGsyn. Of these bp states, only 8OG in Asyn:8OGanti was located in the extrahelical space, whereas the purine bases (A and 8OG) in the other bp states remained inside the DNA helix. Also, free energy landscapes showed that the isomerization processes connecting these four bp states proceeded mostly in the intrahelical space via successive single glycosidic bond rotations of either A or 8OG.

Cell-free fetal DNA (cffDNA) extraction from whole blood by using a fully automatic centrifugal microfluidic device based on displacement of magnetic silica beads

The purpose of this research was to design a fully automated centrifugal microfluidic system (Lab-on-a-Disk) for isolating cell free fetal DNAs (cffDNAs) from whole blood. To achieve this goal, magnetic silica beads were used, such that after attaching cffDNA to them, they were transferred between chambers by using external fixed magnets. All the standards and required steps for cffDNA extraction including plasma separation, adding proteinase K, lysis buffer, binding buffer, washing buffer, and elution buffer were considered in this designed disk. To evaluate the function of the disk, the collected samples were tested from several aspects. First, the purity of extracted plasma from whole blood was investigated which included hemoglobin test, hemocytometer, etc. This disk could extract 1.3 mL pure plasma from 3 mL blood with 45% hematocrit. The results of the extracted plasma showed 99% purity. Finally, the cffDNAs were examined by using a male fetal gender identification kit and real-time PCR machine. The results indicated the correct function of the disk in extracting cffDNAs in samples of 10 Landa from cycle 34 onwards. Compared to the clinical method, the disk not only was able to extract cffDNA in 20 min but also it led to less buffer consumption since the disk only required 1 mL plasma for extraction of cffDNAs.

THE PREFERENCE PRIORITY OF Bacillus subtilis IN UPTAKING FREE DNA DURING THE NATURAL TRANSFORMATION

Although genetic material is vertically transferred between generations via sexual or asexual reproduction, similarities in some chromosome and gene parts of unrelated organisms provide important clues for another way of transfer. The mobility of genetic information among different organisms, known as horizontal gene transfer (HGT) has immediate or delayed effects on the recipient host. One of the most notable mechanisms of HGT is natural transformation (NT), a process in which cells take free DNA from the extracellular environment and incorporate it into their chromosomes by homologous recombination. NT is widely conserved in many bacterial species as it can promote to spread of resistance genes. Although it is known that many organisms rely on HGT, there is limited information about how they decide which particular genetic material to horizontally transfer. Here, I have investigated the preference priority among different gene sources presented under certain stress conditions for Bacillus subtilis possessing NT ability. To test this, two DNA specimens (E and C) with different sequence contents of the same length were presented to B. subtilis under different stress environments (BK, BC, BE and BCE). The hypothesis was evaluated according to the analysis of the results of colonial formations on selective plates (pE, pC and pCE). The obtained data presented a strong positive correlation that the bacteria have preference priority during NT depending on a stimulator. The tendency of the bacteria to uptake useful DNA fragments in a specific environment can be suggested. For instance, the majority of colonies grow on pE plates rather than the pC and pCE when the transformation media includes erythromycin (Eryt) as an inducer. Although the data significantly overlaps with the idea claiming that the bacteria have a preference priority to uptake free DNAs during NT, further investigations are needed to support the present data and for better understanding of the phenomenon.

Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation.

The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined the crystal structures of recombinant human p52 protein in complex with a P-selectin(PSel)-κB DNA (5'-GGGGTGACCCC-3') (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Microsecond molecular dynamics (MD) simulations of free DNAs and p52 bound complexes reveal that free DNAs exhibit distinct preferred conformations, and p52:p52 homodimer induces the least amount of DNA conformational changes when bound to the more transcriptionally active natural G/C-centric PSel-κB, but adopts closed conformation when bound to the mutant A/T and swap DNAs due to their narrowed minor grooves. Our binding assays further demonstrate that the fast kinetics favored by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and pinpoint the importance of binding kinetics, dictated by DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.

Impact of 5HydroxyMethylCytosine (5hmC) on reverse/direct association of cell-cycle, apoptosis, and extracellular matrix pathways in gastrointestinal cancers

BackgroundAberrant levels of 5-hydroxymethylcytosine (5-hmC) can lead to cancer progression. Identification of 5-hmC-related biological pathways in cancer studies can produce better understanding of gastrointestinal (GI) cancers.We conducted a network-based analysis on 5-hmC levels extracted from circulating free DNAs (cfDNA) in GI cancers including colon, gastric, and pancreatic cancers, and from healthy donors. The co-5-hmC network was reconstructed using the weighted-gene co-expression network method. The cancer-related modules/subnetworks were detected. Preservation of three detected 5-hmC-related modules was assessed in an external dataset. The 5-hmC-related modules were functionally enriched, and biological pathways were identified. The relationship between modules was assessed using the Pearson correlation coefficient (p-value < 0.05). An elastic network classifier was used to assess the potential of the 5-hmC modules in distinguishing cancer patients from healthy individuals. To assess the efficiency of the model, the Area Under the Curve (AUC) was computed using five-fold cross-validation in an external dataset. ResultsThe main biological pathways were the cell cycle, apoptosis, and extracellular matrix (ECM) organization. Direct association between the cell cycle and apoptosis, inverse association between apoptosis and ECM organization, and inverse association between the cell cycle and ECM organization were detected for the 5-hmC modules in GI cancers. An AUC of 92% (0.73–1.00) was observed for the predictive model including 11 genes.ConclusionThe intricate association between biological pathways of identified modules may reveal the hidden significance of 5-hmC in GI cancers. The identified predictive model and new biomarkers may be beneficial in cancer detection and precision medicine using liquid biopsy in the early stages.

Gastric Cancer Diagnosis: From Imaging Techniques to Biochemical Biomarkers.

Gastric cancer (GC) has been found to be the second leading cause of cancer-associated deaths in the world, and is usually detected in the advanced stages. It has been shown that surgery is the major therapeutic approach in the treatment of subjects with GC. Hence, early and fast diagnosis of this malignancy is very important for good subject outcomes. Non-invasive diagnostic platforms, including evolutionary endoscopy and positron emission tomography (PET), are employed for the diagnosis of subjects with GC. Along with imaging techniques, the utilization of biomarkers has emerged as a new diagnosis option for early and fast detection of GC. Multiple lines of evidence have revealed a variety of biomarkers, including microRNAs, exosomes, circulating tumor cells, circular RNAs, cell free DNAs, and various proteins, which could be used as diagnostic biomarkers in patients with GC. Taken together, these findings suggest that the joint application of imaging techniques and different biomarkers could be introduced as a new detection approach in the treatment and screening response to therapy in the subjects with GC. Herein, we have summarized various imaging techniques and biomarkers as powerful tools in the detection of GC.

Cell free bacterial DNAs in human plasma provide fingerprints for immune-related diseases

Microbiota is increasingly being recognized as an important factor in human health. Alterations in the bacterial community structure have been implicated in a wide range of human diseases. Most therapeutic strategies targeting microbiota, such as probiotics, prebiotics and fecal transplantation, aim to modulate the bacterial community. Herein we demonstrate that the cell free bacterial DNAs (cfbDNAs) are extensively observed in human bloodstreams and gradually arise along with growth. Moreover, patients with immune-related diseases, e.g. inflammatory bowel disease (IBD), kawasaki disease (KD) and HIV, own higher amounts of cfbDNAs with lower microbial biodiversity compared with healthy individuals by using real-time PCR measurement and high-throughput 16S rDNA sequences analyses. Further comparisons reveal that 173 genera exhibit preferential abundance in either healthy individuals or patients, providing “molecular phenotypes” of the corresponding diseases in a manner of microbiome, especially in those IBD patients post/pre-therapy. Although the origination and function of these cfbDNAs in human circulating bloodstreams remain unclear, their taxonomic variations offer fingerprints for potential applications in noninvasive and safe pre-diagnosis or prognosis, and provide clues for further understanding of the host-microbiota interactions.

Intracellular Nonenzymatic In Situ Growth of Three-Dimensional DNA Nanostructures for Imaging Specific Biomolecules in Living Cells.

Real-time in situ monitoring of low-abundance cancer biomarkers (e.g., miRNAs and proteins) in living cells by nonenzymatic assembly entirely from original DNA probes remains unexplored due to an extremely complex intracellular environment. Herein, a nonenzymatic palindrome-catalyzed DNA assembly (NEPA) technique is developed to execute the in situ imaging of intracellular miRNAs by assembling a three-dimensional nanoscale DNA spherical structure (NS) with low mobility from three free hairpin-type DNAs rather than from DNA intermediates based on the interaction of designed terminal palindromes. Target miRNA was detected down to 1.4 pM, and its family members were distinguished with almost 100% accuracy. The subcellular localization of NS products can be visualized in real time. The NEPA-based sensing strategy is also suitable for the intracellular in situ fluorescence imaging of cancer-related protein receptors, offering valuable insight into developing sensing protocols for understanding the biological function of vital biomolecules in disease pathogenesis and future therapeutic applications.

Cell Free DNA and Genometastasis

Cell free DNAs (cfDNA) are short DNA fragments which are present in all biological fluids and cell culture medium. They were first detected in blood plasma by Mandel and Metais in 1948. cfDNAs are mostly endogenous-derived fragments that are determined in lipid/protein rich complexes or particles with membranes. In healthy individuals, there are small amounts of mono-nucleosome forms of cfDNA in the peripheral circulation. cfDNA can bind to proteins and phospholipids on cell surfaces. This mechanism may related to absorbance and release of cfDNA. Different enzymes such as deoxyribonuclease (DNase) may facilitate the unbounding and recirculation of membrane bound cfDNAs. The investigation of cfDNA that circulates freely in the blood initiated its application in clinical research including diagnosis. Especially, cfDNA in mother’s blood, which originated in fetus, has been in widely used in prenatal diagnosis already. Moreover, cfDNA has been applied in much clinical research, including cancer, organ transplantation, auto-immune diseases, trauma, myocardial infarcts, and sepsis. Although it is extremely useful to analyze cfDNA for certain pathologies and physiological conditions, there is no definite information about their fragment dimensions, origins nor their character. In this review, the possible origins of cfDNA are explored with an overview of the literature regarding cfDNA and also, its role in genometastasis has been investigated. In addition, the rapidly increasing diagnostic use in recent years, especially its advantages in prenatal diagnosis are discussed.

A smart fluorescent biosensor for the highly sensitive detection of BRCA1 based on a 3D DNA walker and ESDR cascade amplification.

Nucleic acid analysis plays an important role in the diagnosis of diseases. There is a continuous demand to develop rapid and sensitive methods for the specific detection of nucleic acids. Herein, we constructed a highly sensitive and rapid fluorescent biosensor for the detection of BRCA1 by coupling a 3D DNA walker machine with spontaneous entropy-driven strand displacement reactions (ESDRs). In this study, the 3D DNA walker machine was well activated by the target DNA; this resulted in the cyclic utilization of the target DNA and the release of intermediate DNAs. Subsequently, the free intermediate DNAs triggered the circulation process of ESDRs with the help of the assistant probe A, leading to a significant enhancement of the fluorescence intensity. Due to the robust execution of the 3D DNA walker machine and highly efficient amplification capability of ESDRs, the developed biosensing method shows a wide linear range from 0.1 pM to 10 nM with the detection limit as low as 41.44 fM (S/N = 3). Moreover, the constructed biosensor displays superior specificity and has been applied to monitor BRCA1 in complex matrices. Thus, this elaborated cascade amplification biosensing strategy provides a potential platform for the bioassays of nucleic acids and the clinical diagnosis of diseases.

Plasma epidermal growth factor receptor mutation testing with a chip-based digital PCR system in patients with advanced non-small cell lung cancer

ObjectivesEpidermal growth factor receptor (EGFR) mutation testing is a companion diagnostic to determine eligibility for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). Recently, plasma-based EGFR testing by digital polymerase chain reaction (dPCR), which enables accurate quantification of target DNA, has shown promise as a minimally invasive diagnostic. Here, we aimed to evaluate the accuracy of a plasma-based EGFR mutation test developed using chip-based dPCR-based detection of 3 EGFR mutations (exon 19 deletions, L858R in exon 21, and T790M in exon 20). Materials and methodsForty-nine patients with NSCLC harboring EGFR-activating mutations were enrolled, and circulating free DNAs (cfDNAs) were extracted from the plasma of 21 and 28 patients before treatment and after progression following EGFR-TKI treatment, respectively. ResultsUsing reference genomic DNA containing each mutation, the detection limit of each assay was determined to be 0.1%. The sensitivity and specificity of detecting exon 19 deletions and L858R mutations, calculated by comparing the mutation status in the corresponding tumors, were 70.6% and 93.3%, and 66.7% and 100%, respectively, showing similar results compared with previous studies. T790M was detected in 43% of 28 cfDNAs after progression with EGFR-TKI treatment, but in no cfDNAs before the start of the treatment. ConclusionThis chip-based dPCR assay can facilitate detection of EGFR mutations in cfDNA as a minimally invasive method in clinical settings.

Endothelial Repair and Endothelial Cell-Derived Secretome

Growing evidence supports the hypothesis that endothelial cell-derived microparticles (MPs) might contribute to the pathogenesis of cardiovascular (CV) disease. Endothelial cell-derived MPs play a pivotal role in the regulation of the endogenous repair system, thrombosis, coagulation, inflammation, immunity and metabolic memory phenomenon. There is evidence that the MPs are secreted actively accompanied to other regulatory molecules. All these actively synthetizing and secreting factors include proteins, adhesion and intercellular signal molecules, peptides, lipids, free DNAs, microRNAs, and even microparticles (MPs) are defined as cellular secretome. The proteomic profile of secretome is under tightly control of genetic and epigenetic mechanisms, which may altered a secretion of the proteins involved into MPs’ organization. Finally, this may contribute the modification of MP’s after their secretion and throughout transfer to the target cells. As a result, communicative ability of endothelial cell-derived MPs may sufficiently worse. Subsequently, cross talk between some components of secretome might modulate delivering cargos of MPs and their regenerative and proliferative capabilities via intercellular signaling networks. The aim of the review is to discuss the effect of various components of secretome on MP-dependent effects on endothelium.

The Endothelial Cell Secretome as a Factor of Endothelium Reparation: The Role of Microparticles

The secretome is considered a combination of factors produced by cells due to abundant spectrum of autocrine/ paracrine triggers. All these actively synthetizing and secreting factors include proteins, adhesion and intercellular signal molecules, peptides, lipids, free DNAs, microRNAs, and microparticles (MPs). The components of secretome mutually may interact and thereby modify the MPs’ structure and functionality. As a result, communicative ability of endothelial cell derived MPs may sufficiently impaire. Subsequently, cross talk between some components of secretome might modulate delivering cargos of MPs and their regenerative and proliferative capabilities via intercellular signaling networks. The aim of the review is to discuss the effect of various components of secretome on MP-dependent effects on endothelium.

Lamellar cationic lipid-DNA complexes from lipids with a strong preference for planar geometry: A Minimal Electrostatic Model.

We formulate and analyze a minimal model, based on condensation theory, of the lamellar cationic lipid (CL)-DNA complex of alternately charged lipid bilayers and DNA monolayers in a salt solution. Each lipid bilayer, composed by a random mixture of cationic and neutral lipids, is assumed to be a rigid uniformly charged plane. Each DNA monolayer, located between two lipid bilayers, is formed by the same number of parallel DNAs with a uniform separation distance. For the electrostatic calculation, the model lipoplex is collapsed to a single plane with charge density equal to the net lipid and DNA charge. The free energy difference between the lamellar lipoplex and a reference state of the same number of free lipid bilayers and free DNAs, is calculated as a function of the fraction of CLs, of the ratio of the number of CL charges to the number of negative charges of the DNA phosphates, and of the total number of planes. At the isoelectric point the free energy difference is minimal. The complex formation, already favoured by the decrease of the electrostatic charging free energy, is driven further by the free energy gain due to the release of counterions from the DNAs and from the lipid bilayers, if strongly charged. This minimal model compares well with experiment for lipids having a strong preference for planar geometry and with major features of more detailed models of the lipoplex.

Application of PCR Technique in Combination with DNase Treatment for Detection of Viable Lactobacillus acidophilus Bacteria

AbstractIn this study, we examined whether application of DNase I can serve as differential eliminator of DNAs from dead cells, leaving viable probiotic lactic acid bacteria such as Lactobacillus acidophilus to be assessed by polymerase chain reaction (PCR). When dead cells were treated with DNase I, DNA amplification was not completely suppressed. Increasing the concentration of DNase I, up to 66 u/100 μL, and the preparation of dead cells using high temperatures did not seem to make difference in the level of PCR product from the dead bacteria. Assessment of free DNA degradation, when mixed with dead cells, showed that stability of free DNAs or their degradation by DNase I was not affected by presence of the dead cells. In conclusion, we tend to suggest that for using this technique, one should take great deal of caution and that its reliability should be tested for different species independently.Practical ApplicationsLactobacillus acidophilus is one of the most common probiotic bacteria incorporated into food products. To have their health‐promoting properties, consumption of high levels of the viable bacteria is recommended. Meanwhile, access to reliable protocols for assessment of viable bacteria remains elusive. Recent studies have focused on providing differential conditions for clearance of DNA material of the dead cells followed by quantification of the viable bacteria by molecular techniques. Despite the previous reports on application of DNase treatment along with PCR assays, for evaluation of viable harmful food bacteria, our data do not support the notion to be applied for detection of L. acidophilus.

Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe

Electrostatic redox probes interaction has been widely rendered for DNA quantification. We have established a proof-of-principle by using the ruthenium hexaamine molecule [Ru(NH(3))(6)](3+). We have applied this method for real-time electrochemical monitoring of a loop mediated isothermal amplification (LAMP) amplicon of target genes of Escherichia coli and Staphylococcus aureus by square wave voltammetry (SWV). Ruthenium hexaamine interaction with free DNAs in solution without being immobilized onto the biochip surface enabled us to discard the time-consuming overnight probe immobilization step in DNA quantification. We have measured the changes in the cathodic current signals using screen printed low-cost biochips both in the presence and the absence of LAMP amplicons of target DNAs in the solution-phase. By using this novel probe, we successfully carried out the real-time isothermal amplification and detection in less than 30 min for S. aureus and E. coli with a sensitivity up to 30 copies μL(-1) and 20 copies μL(-1), respectively. The cathode peak height of the current was related to the extent of amplicon formation and the amount of introduced template genomic DNA. Importantly, since laborious probe immobilization is not necessary at all, and both the in vitro amplification and real-time monitoring are performed in a single polypropylene tube using a single biochip, this novel approach could avoid all potential cross-contamination in the whole procedure.

Nuclease-resistant DNA aptamer on gold nanoparticles for the simultaneous detection of Pb2+ and Hg2+ in human serum

There has been great progress in the development of functional DNA-based sensors for the detection of metal ions. However, many functional DNAs are vulnerable to hydrolysis by nucleases in human blood. In addition, the detection methods that are based on DNA often exhibit interference due to the high blood concentrations of other ions, such as K+ and Na+. Therefore, we selected highly Pb2+-specific DNA-aptamer sequences based on CD spectroscopy of 4 G-rich DNA sequences and Hg2+-specific T-rich DNA sequences and immobilized them on gold nanoparticles for the simultaneous detection of Pb2+ and Hg2+ in human serum. We used gold nanoparticles because these have a superior fluorescence-quenching efficiency over a broad range of wavelengths compared with other organic quenchers. In addition, gold nanoparticles have a stabilizing effect on the immobilized DNA, which makes it more resistant to degradation by nucleases than free DNA. As a result, even in the presence of DNase, we were able to simultaneously detect Pb2+ and Hg2+ in serum at concentrations as low as 128pM and 121pM, respectively, within 10min. These detection limits for Pb2+ and Hg2+ were 39-fold and 26.4-fold lower, respectively, than the detection limits that were obtained using free DNAs. Given the multi-color-fluorescence quenching capability of the gold nanoparticles and the possibility of developing functional nucleic acids for the detection of other metal ions, this study extends the application of oligonucleotides to a point-of-care detection system for the detection of multiple harmful metal ions in body fluids.

Following the DNA Ligation of a Single Duplex Using Atomic Force Microscopy

Nick-sealing of a single DNA duplex was studied with the use of atomic force microscopy (AFM). To form a nick between a 47 mer DNA and a 24 mer DNA, the complementary 71 mer template DNA immobilized on an AFM tip was hybridized with the 47 mer DNA and brought into contact with the 24 mer DNA on a substrate surface. The AFM tip and substrate surface were modified with dendron molecules to ensure the formation of a single DNA duplex. When a single nick in the DNA duplex was sealed by DNA ligase during a pause, an increase in the unbinding force was observed after the pause. The change from 24.0 ± 4.4 piconewtons (pN) to 62.8 ± 14.6 pN matched well with the resulting DNA length (71 bp). Additionally, a 30 s pause showed a 3-fold higher nick-sealing probability (60%) than a 10 s pause, while the probability did not increase with a 120 s pause. In the presence of free 47 mer DNAs in solution, the single nick-sealing event could be repeated at other positions.