Deoxyribonucleic Acid Molecules Research Articles

Blockade and Staircase Phenomena of Holes in Mesoscopic Scale $\lambda $ -Deoxyribonucleic Acid/SiO2/Si Structure

Coulomb blockade/staircase phenomena of deoxyribonucleic acid (DNA) molecule have been observed so far. Here, we show a non-Coulomb blockade/staircase phenomenon of holes in a mesoscopic scale $\lambda $ -DNA, which serves as a channel on SiO2/Si structure. It is considered that the phenomena are due to the penetration of the wave function in the DNA into the Si source and drain electrodes via the contacts constructed by DNA/allyl glycidyl ether/Si electrodes.

Electronic Characterization of Au/DNA/ITO Metal-Semiconductor-Metal Diode and Its Application as a Radiation Sensor

Deoxyribonucleic acid or DNA molecules expressed as double-stranded (DSS) negatively charged polymer plays a significant role in electronic states of metal/silicon semiconductor structures. Electrical parameters of an Au/DNA/ITO device prepared using self-assembly method was studied by using current–voltage (I-V) characteristic measurements under alpha bombardment at room temperature. The results were analyzed using conventional thermionic emission model, Cheung and Cheung’s method and Norde’s technique to estimate the barrier height, ideality factor, series resistance and Richardson constant of the Au/DNA/ITO structure. Besides demonstrating a strongly rectifying (diode) characteristic, it was also observed that orderly fluctuations occur in various electrical parameters of the Schottky structure. Increasing alpha radiation effectively influences the series resistance, while the barrier height, ideality factor and interface state density parameters respond linearly. Barrier height determined from I–V measurements were calculated at 0.7284 eV for non-radiated, increasing to about 0.7883 eV in 0.036 Gy showing an increase for all doses. We also demonstrate the hypersensitivity phenomena effect by studying the relationship between the series resistance for the three methods, the ideality factor and low-dose radiation. Based on the results, sensitive alpha particle detectors can be realized using Au/DNA/ITO Schottky junction sensor.

Solving the maximal matching problem with DNA molecules in Adleman–Lipton model

The maximal matching problem (MMP) is to find maximal edge subsets in a given undirected graph, that no pair of edges are adjacent in the subsets. It is a vitally important NP-complete problem in graph theory and applied mathematics, having numerous real life applications in optimal combination and linear programming fields. It can be difficultly solved by the electronic computer in exponential level time. Meanwhile in previous studies deoxyribonucleic acid (DNA) molecular operations usually were used to solve NP-complete continuous path search problems, e.g. HPP, traveling salesman problem, rarely for NP-hard problems with discrete vertices or edges solutions, such as the minimum vertex cover problem, graph coloring problem and so on. In this paper, we present a DNA algorithm for solving the MMP with DNA molecular operations. For an undirected graph with [Formula: see text] vertices and [Formula: see text] edges, we reasonably design fixed length DNA strands representing vertices and edges of the graph, take appropriate steps and get the solutions of the MMP in proper length range using [Formula: see text] time. We extend the application of DNA molecular operations and simultaneously simplify the complexity of the computation.

The Application of Pulsed Field Gel Electrophoresis in Clinical Studies.

Pulsed-field gel electrophoresis is a method applied in separating large segments of deoxyribonucleotide using an alternating and cross field. In a uniform magnetic field, components larger than 50kb pass a route through the gel and since the movement of DNA (Deoxyribonucleic acid) molecules are in a Zigzag form, separation of DNAs as bands carried out better via gel. PFGE in microbiology is a standard method which is used for typing of bacteria. It is also a very useful tool in epidemiological studies and gene mapping in microbes and mammalian cell, also motivated development of large-insert cloning system such as bacterial and yeast artifical chromosomes. In this method, close and similar species in terms of genetic patterns show alike profiles regarding DNA separation, and those ones which don't have similarity or are less similar, reveal different separation profiles. So this feature can be used to determine the common species as the prevalence agent of a disease. PFGE can be utilized for monitoring and evaluating different micro-organisms in clinical samples and existing ones in soil and water. This method can also be a reliable and standard method in vaccine preparation. In recent decades, PFGE is highly regarded as a powerful tool in control, prevention and monitoring diseases in different populations.

Polymerase chain reaction in microfluidic devices.

The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.

A Schema-Based DNA Algorithm Applicable on Graphics Processing Units

In this paper, an evolutionary algorithm inspired by biological deoxyribonucleic acid (DNA) computing is proposed to perform the task of optimization. As a novel branch of computational intelligence, DNA computing has the strong computing and proliferating capability to in DNA strands by DNA encoding and matching in the molecule layer. However, it is difficult to apply DNA computing to search processing in a nonlinear hyperspace, because traditional DNA computing often relies on biochemical reactions of DNA molecules and may result in incorrect or undesirable computations. To utilize the advantages and avoid the problems of biological DNA computing, this article proposes an artificial DNA computing approach, which accelerates the process by means of the operation of restriction enzymes based on constructed schemata. After DNA encoding, the typical DNA database is constructed and evolved by evolutionary algorithms and the schema theory of genetic algorithm (GA) integrated to enhance the accuracy and convergence rate of exploration in the search space. Meanwhile, to fully utilize parallelism characteristics of DNA computing, a novel algorithm, named parallel DNA computing algorithm, which is tailored to be implemented in graphics processing unit devices, is proposed and exanimated its performance on benchmark optimization problems. Experimental results demonstrate that the proposed method presents excellent expected processing time efficiency, compared with its counterparts without the designed accelerating processes, such as schemata, restriction enzyme, and migration. As well, the performance is also distinctly superior to two renowned GAs.

A Paradigm Shift in Low Dose Radiation Biology

When ionizing radiation traverses biological material, some energy depositions occur and ionize directly deoxyribonucleic acid (DNA) molecules, the critical target. A classical paradigm in radiobiology is that the deposition of energy in the cell nucleus and the resulting damage to DNA are responsible for the detrimental biological effects of radiation. It is presumed that no radiation effect would be expected in cells that receive no direct radiation exposure through nucleus. The risks of exposure to low dose ionizing radiation are estimated by extrapolating from data obtained after exposure to high dose radiation. However, the validity of using this dose-response model is controversial because evidence accumulated over the past decade has indicated that living organisms, including humans, respond differently to low dose radiation than they do to high dose radiation. Moreover, recent experimental evidences from many laboratories reveal the fact that radiation effects also occur in cells that were not exposed to radiation and in the progeny of irradiated cells at delayed times after radiation exposure where cells do not encounter direct DNA damage. Recently, the classical paradigm in radiobiology has been shifted from the nucleus, specifically the DNA, as the principal target for the biological effects of radiation to cells. The universality of target theory has been challenged by phenomena of radiation-induced genomic instability, bystander effect and adaptive response. The new radiation biology paradigm would cover both targeted and non-targeted effects of ionizing radiation. The mechanisms underlying these responses involve biochemical/molecular signals that respond to targeted and non-targeted events. These results brought in understanding that the biological response to low dose radiation at tissue or organism level is a complex process of integrated response of cellular targets as well as extra-cellular factors. Biological understanding of the effects of radiation can be used to improve the assessment of low dose radiation risk. In this article, the mechanisms of targeted and non-targeted responses, and interrelation between the phenomena on cellular injury after exposure to low doses of radiation as they relate to low dose radiation effects will be reviewed. Received:14 October 2014; Revised:1 April 2015; Accepted: 14 April 2015

DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.

Local piezoresponse and polarization switching in nucleobase thymine microcrystals

Thymine (2-oxy-4-oxy-5 methyl pyrimidine) is one of the four nucleobases of deoxyribonucleic acid (DNA). In the DNA molecule, thymine binds to adenine via two hydrogen bonds, thus stabilizing the nucleic acid structure and is involved in pairing and replication. Here, we show that synthetic thymine microcrystals grown from the solution exhibit local piezoelectricity and apparent ferroelectricity, as evidenced by nanoscale electromechanical measurements via Piezoresponse Force Microscopy. Our experimental results demonstrate significant electromechanical activity and polarization switchability of thymine, thus opening a pathway for piezoelectric and ferroelectric-based applications of thymine and, perhaps, of other DNA nucleobase materials. The results are supported by molecular modeling of polarization switching under an external electric field.

Isolation of thermally sensitive protein-binding oligonucleotides on a microchip.

This paper presents a microfluidic chip capable of isolating thermally sensitive protein-binding aptamer candidates. The chip makes use of bead-immobilized target molecules and DNA (deoxyribonucleic acid) sequences to enable a simplified chip design, in which affinity selection and PCR (polymerase chain reaction) amplification of target-binding sequences occur in temperature-controlled microchambers. Using pressure-driven flow, buffer containing single-stranded DNA molecules with randomized sequences is cycled through a series of affinity selection and PCR amplification steps on microbeads. Successive introduction of the sample to each chamber effects a process of competition whereby DNA strands with weak binding strength to target molecules are rejected in favor of strongly binding sequences. Using bead-based PCR, the amplification step was miniaturized and integrated with affinity selection, resulting in significant reductions in process time and reagent use. As a demonstration, temperature-dependent selection and amplification of single-stranded oligonucleotides that bind to human Immuno-globulin E (IgE) was performed in 4 h, a 20-fold reduction in process time as compared to conventional methods that would require approximately a week. Fluorescent binding assays then demonstrated that the desired temperature specificity was imparted to the aptamer candidates within just one round of selection, and within two rounds the aptamer candidates exhibited enhanced affinity toward IgE.

Field-effect transistor with a chemically synthesized MoS2 sensing channel for label-free and highly sensitive electrical detection of DNA hybridization

A field-effect transistor (FET) with two-dimensional (2D) few-layer MoS2 as a sensing-channel material was investigated for label-free electrical detection of the hybridization of deoxyribonucleic acid (DNA) molecules. The high-quality MoS2-channel pattern was selectively formedthrough the chemical reaction of the Mo layer with H2S gas. The MoS2 FET was very stable in an electrolyte and inert to pH changes due to the lack of oxygen-containing functionalities on the MoS2 surface. Hybridization of single-stranded target DNA molecules with single-stranded probe DNA molecules physically adsorbed on the MoS2 channel resulted in a shift of the threshold voltage (V th) in the negative direction and an increase in the drain current. The negative shift in V th is attributed to electrostatic gating effects induced by the detachment of negatively charged probe DNA molecules from the channel surface after hybridization. A detection limit of 10 fM, high sensitivity of 17 mV/dec, and high dynamic range of 106 were achieved. The results showed that a bio-FET with an ultrathin 2D MoS2 channel can be used to detect very small concentrations of target DNA molecules specifically hybridized with the probe DNA molecules.

A label-free biosensor based on organic transistors by using the interaction of mercapto DNA and gold electrodes

Organic thin-film transistors (OTFTs) with Au electrodes were successfully used as transducers for label-free deoxyribonucleic acid (DNA) sensors. Single-strand DNA (ssDNA), perfectly-matched double-strand DNA (dsDNA) and mis-matched DNA were immobilized on the surface of the source/drain electrodes of three OTFT devices respectively. The ssDNA molecules with mercapto group (–SH) can be well immobilized on the surface of Au electrode by chemical bond between –SH and Au atom. According to the significant difference in channel current, which was attributed to the changed contact resistances by introducing different DNA molecules on Au electrode, ssDNA, matched-dsDNA and mismatched-dsDNA were differentiated successfully in the experiments. The results may provide a promising approach for detecting DNA specificity and hybridization with label-free.

Concentration-dependent effects on fully hydrated DNA at terahertz frequencies.

Using terahertz time-domain spectroscopy (THz-TDS), the frequency-dependent dielectric constant of deoxyribonucleic acid (DNA) in solution was measured. The response of the buffer solution is dominated by two Debye modes in this frequency range, and, from an analysis of the concentration dependence, the presence of the DNA increases the main relaxation time and dielectric constant. This reflects the fact that the water in the hydration layer is more tightly bound under the influence of the DNA molecule in comparison to bulk water. This dynamical slowing down with increasing DNA concentration is similar to what is observed with purine nucleotides, but opposite to the behavior of pyrimidine nucleotides. In addition, a suspension model was used with the concentration-dependent data to isolate the dielectric response of the hydrated DNA molecule. The data for the hydrated DNA molecule is still dominated by a Debye response. It is also possible to determine the thickness of the hydration layer, and the DNA molecule influences the surrounding water out to 16 or 17 Å, which corresponds to about six effective hydration layers.

The DNA Dynamics Near Nanopores at Sub-Millisecond and Sub-Micrometer Levels by the Ultraviolet Light Spot

Phenomenon of deoxyribonucleic acid (DNA) translocation through a nanopore is a significant interest in the field of biophysics. In the condition of applied voltage, coiled DNA molecules in the electrolyte approach to the mouth of a nanopore and thread into a nanopore by uncoiling its structure. Then DNA molecules recoil and diffuse outward from a nanopore after DNA translocation. Because DNA molecules uncoil to thread into a nanopore and recoil after DNA translocation, DNA dynamics near a nanopore strongly correlate with DNA translocation. Here, we propose an optical method for investigating DNA dynamics near nanopores at sub-millisecond and sub-micrometer levels by using ultraviolet light and silicon nanopores. Silicon nanopores, which have high refractive index and extinction coefficient at ultraviolet light, induces the light spot, whose size is 100nm, upon nanopores due to low transmitted light condition. By threading into nanopores, DAPI-stained DNA molecules pass through the ultraviolet light spot and emit fluorescence. We use the fluorescence intensity trace as the information of DNA translocation through nanopores. As the results of our measurement, the fluorescence intensity traces were in accordance with previous investigation of the voltage dependence of DNA electrophoresis mobility. Furthermore, our analytical results of fluorescence intensity traces showed the correlation between DNA conformations during DNA translocation through nanopores, which are unfolded or folded DNA translocation, and DNA dynamics after DNA translocation through nanopores, which reveal the usefulness of our optical method for investigating DNA dynamics near nanopores.

Social behavior in bacterial nanonetworks: challenges and opportunities

Molecular communication promises to enable communication between nanomachines with a view to increasing their functionalities and open up new possible applications. Due to some of the biological properties, bacteria have been proposed as a possible information carrier for molecular communication, and the corresponding communication networks are known as \textit{bacterial nanonetworks}. The biological properties include the ability for bacteria to mobilize between locations and carry the information encoded in Deoxyribonucleic Acid (DNA) molecules. However, similar to most organisms, bacteria have complex social properties that govern their colony. These social characteristics enable the bacteria to evolve through various fluctuating environmental conditions by utilizing cooperative and non-cooperative behaviors. This article provides an overview of the different types of cooperative and non-cooperative social behavior of bacteria. The challenges (due to non-cooperation) and the opportunities (due to cooperation) these behaviors can bring to the reliability of communication in bacterial nanonetworks are also discussed. Finally, simulation results on the impact of bacterial cooperative social behavior on the end-to-end reliability of a single-link bacterial nanonetwork are presented. The article concludes with highlighting the potential future research opportunities in this emerging field.

Theoretical study of the transpore velocity control of single-stranded DNA.

The electrokinetic transport dynamics of deoxyribonucleic acid (DNA) molecules have recently attracted significant attention in various fields of research. Our group is interested in the detailed examination of the behavior of DNA when confined in micro/nanofluidic channels. In the present study, the translocation mechanism of a DNA-like polymer chain in a nanofluidic channel was investigated using Langevin dynamics simulations. A coarse-grained bead-spring model was developed to simulate the dynamics of a long polymer chain passing through a rectangular cross-section nanopore embedded in a nanochannel, under the influence of a nonuniform electric field. Varying the cross-sectional area of the nanopore was found to allow optimization of the translocation process through modification of the electric field in the flow channel, since a drastic drop in the electric potential at the nanopore was induced by changing the cross-section. Furthermore, the configuration of the polymer chain in the nanopore was observed to determine its translocation velocity. The competition between the strength of the electric field and confinement in the small pore produces various transport mechanisms and the results of this study thus represent a means of optimizing the design of nanofluidic devices for single molecule detection.

Introduction

1. Deborah Josko, Ph.D. MLT(ASCP)M, SM[⇑][1] 1. Associate Professor and Program Director, Department of Clinical Laboratory Sciences, Medical Laboratory Science Program, Rutgers, The State University of New Jersey – School of Health Related Professions, Scotch Plains, NJ <!-- --> 1. Address for correspondence: Deborah Josko, Ph.D. MLT(ASCP)M, SM, Associate Professor, Department of Clinical Laboratory Sciences, Medical Laboratory Science Program, Rutgers, The State University of New Jersey – School of Health Related Professions 1776 Raritan Road, Scotch Plains, NJ 07076. (908) 889-2422. daj100{at}shrp.rutgers.edu. 1. Discuss the history of DNA sequencing. 2. Describe the components of DNA and DNA synthesis. 3. Compare and contrast dNTPs and ddNTPs. 4. List several next generation sequencing platforms. In 1953, James Watson and Francis Crick were credited with discovering the structure of the DNA molecule: a three dimensional, double helical complex which dictates the genetic code for all forms of life.1 Although much controversy exists over this discovery notably that the model Watson and Crick proposed was the compilation of the work of several pioneers before them,2 they were awarded the Noble Prize in Physiology or Medicine in 1962 along with Maurice Hugh Frederick Wilkins “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.”3 The DNA molecule consists of a sugar (deoxyribose) and phosphate backbone along with four chemical base pairs: adenine (A), thymine (T), cytosine (C), and guanine (G). Each base pair is covalently linked to the sugar phosphate backbone. The double helical structure forms once each base pair links to the complementary base on the corresponding strand of deoxyribonucleic acid (DNA) through hydrogen bonding. Due to the chemical make-up of each base, adenine always binds with thymine while cytosine always binds with guanine. The base, sugar and phosphate together constitute a nucleotide.4 A key property of the DNA molecule is that it can replicate itself and synthesize a new copy of DNA from the preexisting strand during cell division. DNA synthesis requires several components: DNA template; primer; deoxyribonucleotide triphosphates (dNTPs): deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP); various enzymes; and an energy source to fuel the reaction adenosine triphosphate (ATP).5 ABBREVIATIONS : A - adenine; ATP - adenosine triphosphate; C - cytosine; dATP - deoxyadenosine triphosphate; dCTP - deoxycytidine triphosphate; ddNTP - dideoxynucleotide triphosphate; dGTP - deoxyguanosine triphosphate; DNA - deoxyribonucleic acid; dNTP - deoxynucleotide triphosphate; dTTP - deoxythymidine triphosphate; G - guanine; mRNA - messenger ribonucleic acid; NGS – next generation sequencing; OH - hydroxyl; SMRT - single molecule real time; T - thymine. 1. Discuss the history of DNA sequencing. 2. Describe the components of DNA and DNA synthesis. 3. Compare and contrast dNTPs and ddNTPs. 4. List several next generation sequencing platforms. [1]: #corresp-1

Genomic instantiation of consciousness in neurons through a biophoton field theory.

A theoretical framework is developed based on the premise that brains evolved into sufficiently complex adaptive systems capable of instantiating genomic consciousness through self-awareness and complex interactions that recognize qualitatively the controlling factors of biological processes. Furthermore, our hypothesis assumes that the collective interactions in neurons yield macroergic effects, which can produce sufficiently strong electric energy fields for electronic excitations to take place on the surface of endogenous structures via alpha-helical integral proteins as electro-solitons. Specifically the process of radiative relaxation of the electro-solitons allows for the transfer of energy via interactions with deoxyribonucleic acid (DNA) molecules to induce conformational changes in DNA molecules producing an ultra weak non-thermal spontaneous emission of coherent biophotons through a quantum effect. The instantiation of coherent biophotons confined in spaces of DNA molecules guides the biophoton field to be instantaneously conducted along the axonal and neuronal arbors and in-between neurons and throughout the cerebral cortex (cortico-thalamic system) and subcortical areas (e.g., midbrain and hindbrain). Thus providing an informational character of the electric coherence of the brain - referred to as quantum coherence. The biophoton field is realized as a conscious field upon the re-absorption of biophotons by exciplex states of DNA molecules. Such quantum phenomenon brings about self-awareness and enables objectivity to have access to subjectivity in the unconscious. As such, subjective experiences can be recalled to consciousness as subjective conscious experiences or qualia through co-operative interactions between exciplex states of DNA molecules and biophotons leading to metabolic activity and energy transfer across proteins as a result of protein-ligand binding during protein-protein communication. The biophoton field as a conscious field is attributable to the resultant effect of specifying qualia from the metabolic energy field that is transported in macromolecular proteins throughout specific networks of neurons that are constantly transforming into more stable associable representations as molecular solitons. The metastability of subjective experiences based on resonant dynamics occurs when bottom-up patterns of neocortical excitatory activity are matched with top-down expectations as adaptive dynamic pressures. These dynamics of on-going activity patterns influenced by the environment and selected as the preferred subjective experience in terms of a functional field through functional interactions and biological laws are realized as subjectivity and actualized through functional integration as qualia. It is concluded that interactionism and not information processing is the key in understanding how consciousness bridges the explanatory gap between subjective experiences and their neural correlates in the transcendental brain.

Refluxing synthesis, photoluminescence and binding ability to deoxyribonucleic acid of water-soluble rare earth ion-doped LaF3 nanoparticles.

Water-soluble rare earth ion (Ce3+, Tb3+)-doped LaF3 nanoparticles with the ability to bind to deoxyribonucleic acid (DNA) were prepared by the refluxing method in a glycerol/water mixture and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), photoluminescence (PL) spectra, and so on. The obtained LaF3:Ce3+, LaF3:Tb3+ and LaF3:Ce3+, Tb3+ nanoparticles are well crystallized with a hexagonal structure and composed of spherical particles with an average size from 11 to 19 nm. The as-prepared samples can be dispersed into water to form a colloidal solution. Under ultraviolet (UV) light irradiation, the emission lines of Tb3+ in the co-doped LaF3:Ce3+, Tb3+ are evidently strengthened compared to those in the single-doped LaF3:Tb3+ nanoparticles, which is due to the energy transfer from Ce3+ to Tb3+ ions in the LaF3:Ce3+, Tb3+ samples. The biological experiment confirms that the water-soluble LaF3:Ce3+, Tb3+ nanoparticles can be bonded to the DNA molecules and emit visible light under UV irradiation. These luminescent nanoparticles could be used similarly to ethidium bromide (EtBr), which has been used extensively as a DNA staining reagent. The advantage that LaF3:Ce3+, Tb3+ nanoparticles have lower toxicity than EtBr makes them a potential reagent instead of EtBr in the DNA staining in biological experiments.

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.