Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

The unique ability to rapidly amplify low copy number DNA has made in vitro Polymerase Chain Reaction one of the most fundamental techniques in modern biology. In order to harness this technique to its full potential, certain obstacles such as nonspecific by-products, low yield and complexity of GC rich and long genomic DNA amplification need to be surmounted. As in vitro PCR does not have any regulatory mechanisms unlike its counterpart in vivo DNA replication machinery, scientists often use a number of additives like glycerol, betaine, dimethyl sulphoxide and formamide in order to achieve the perfection of in vivo systems. In the last two decades nanotechnology has provided excellent solutions to many classical problems in various scientific fields including biotechnology and recently the PCR technique has begun to benefit from this so called “Nano Era”. In this review, the impacts of several nanomaterials on PCR efficiency, specificity and fidelity are described in accordance with the recent literature. Putative interaction mechanisms between nanomaterials and primary PCR components are also addressed in a comprehensive manner

Molecular organization and comparative analysis of chromosome 5B of the wild wheat ancestor Triticum dicoccoides

Wild emmer wheat, Triticum turgidum ssp. dicoccoides is the wild relative of Triticum turgidum, the progenitor of durum and bread wheat, and maintains a rich allelic diversity among its wild populations. The lack of adequate genetic and genomic resources, however, restricts its exploitation in wheat improvement. Here, we report next-generation sequencing of the flow-sorted chromosome 5B of T. dicoccoides to shed light into its genome structure, function and organization by exploring the repetitive elements, protein-encoding genes and putative microRNA and tRNA coding sequences. Comparative analyses with its counterparts in modern and wild wheats suggest clues into the B-genome evolution. Syntenic relationships of chromosome 5B with the model grasses can facilitate further efforts for fine-mapping of traits of interest. Mapping of 5B sequences onto the root transcriptomes of two additional T. dicoccoides genotypes, with contrasting drought tolerances, revealed several thousands of single nucleotide polymorphisms, of which 584 shared polymorphisms on 228 transcripts were specific to the drought-tolerant genotype. To our knowledge, this study presents the largest genomics resource currently available for T. dicoccoides, which, we believe, will encourage the exploitation of its genetic and genomic potential for wheat improvement to meet the increasing demand to feed the world

REAL-TIME WATER QUALITY MONITORING OF AN ARTIFICIAL LAKE USING A PORTABLE, AFFORDABLE, SIMPLE, ARDUINO-BASED OPEN SOURCE SENSOR

Water quality assessment is vital to identify existing problems and any changes that emerge in water sources over a period of time. Conventional water quality monitoring systems remain to be limited to on-site sample collection and further analysis in environmental laboratories. The progress in Arduino-based low-cost and open-source hardware has paved the way for the development of low-cost, portable, and on-site measuring platforms. In this work, we have assembled an Arduino-based open-source water testing platform out of commercially available sensors and controllers. The water testing system was powered by a 9 V battery and had the capability of measuring water turbidity, acidity, and temperature on-site in real-time. The calibration and validation studies were carried out to assess the measurement capabilities of turbidity and pH sensors in the lab using calibration samples and UV-Vis-NIR absorption spectroscopy. The water quality platform was tested in an artificial lake that is located at Sabanci University Campus (Istanbul, Turkey), which serves as a reservoir for treated wastewaters and rainwater. Untreated wastewater samples were collected from the wastewater treatment station of the university for comparison. The measurements performed on several locations along the coast of the artificial lake were also validated in the laboratory. The water testing platform showed significant potential for miniaturization and portability of such analytical platforms for on-site environmental monitoring

Evaluation of the ICT Tuberculosis test for the routine diagnosis of tuberculosis

BACKGROUND: Rapid and accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of infectious cases and thus to reduce its spread. To improve the diagnosis of TB, more rapid diagnostic techniques such as antibody detection methods including enzyme-linked immunosorbent assay (ELISA)-based serological tests and immunochromatographic methods were developed. This study was designed to evaluate the validity of an immunochromatographic assay, ICT Tuberculosis test for the serologic diagnosis of TB in Antalya, Turkey. METHODS: Sera from 72 patients with active pulmonary (53 smear-positive and 19 smear-negative cases) and eight extrapulmonary (6 smear-positive and 2 smear-negative cases) TB, and 54 controls from different outpatient clinics with similar demographic characteristics as patients were tested by ICT Tuberculosis test. RESULTS: The sensitivity, specificity, and negative predictive value of the ICT Tuberculosis test for pulmonary TB were 33.3%, 100%, and 52.9%, respectively. Smear-positive pulmonary TB patients showed a higher positivity rate for antibodies than smear-negative patients, but the difference was not statistically significant. Of the eight patients with extrapulmonary TB, antibody was detected in four patients. CONCLUSION: Our results suggest that ICT Tuberculosis test can be used to aid TB diagnosis in smear-positive patients until the culture results are available

Dispersion quality of amine functionalized multiwall carbon nanotubes plays critical roles in PCR enhancement

Impact of dispersion quality of NH2-MWCNTs (13-18 nm in diameter with a length between 1 and 12 mu m, >99 % purity) in the amplification efficiency of a random DNA oligonucleotide library (96 bp) was investigated. Amplification yield in the presence of non-filtered NH2-MWCNT dispersion, filtered NH2-MWCNT dispersion and surface-attached NH2-MWCNTs was explored, and physical interactions between NH2-MWCNTs and major PCR reagents including DNA template, wild type Taq DNA polymerase enzyme and primers were determined using high resolution polyacrylamide gel electrophoresis, dynamic light scattering, UV-Vis-NIR spectroscopy and scanning electron microscopy techniques. The results revealed that presence of NH2-MWCNT dispersion which was sonicated, centrifuged and filtered, enhanced the total PCR efficiency up to 70 % while the presence of NH2-MWCNT only centrifuged after sonication, inhibited the reaction significantly at similar concentrations. Furthermore, the NH2-MWCNTs coupled covalently onto magnetic microspheres, contributed for the specificity enhancement whilst decreasing the amplification efficiency by 30 % at the maximum concentration, which suggests a removable enhancement system for sensitive applications. On the other hand, the relative hydrodynamic size distribution measurements displayed a clear difference between the filtered NH2 and non-filtered NH2-MWCNT water dispersions, which justifies the inhibition of the amplification by the non-filtered NH2-MWCNTs containing big agglomerates and bundles. Finally, we demonstrated that major PCR components adsorb onto the NH2-MWCNTs with diverse affinities, and maintain their functions after adsorption, which provides a good framework to further develop tunable NH2-MWCNT-carriers to be utilized in various nanobiotechnology and material science applications

Dispersion quality of amine functionalized multiwall carbon nanotubes plays critical roles in polymerase chain reaction enhancement

Impact of dispersion quality of NH2-MWCNTs (13-18 nm in diameter with a length between 1 and 12 mu m, >99 % purity) in the amplification efficiency of a random DNA oligonucleotide library (96 bp) was investigated. Amplification yield in the presence of non-filtered NH2-MWCNT dispersion, filtered NH2-MWCNT dispersion and surface-attached NH2-MWCNTs was explored, and physical interactions between NH2-MWCNTs and major PCR reagents including DNA template, wild type Taq DNA polymerase enzyme and primers were determined using high resolution polyacrylamide gel electrophoresis, dynamic light scattering, UV-Vis-NIR spectroscopy and scanning electron microscopy techniques. The results revealed that presence of NH2-MWCNT dispersion which was sonicated, centrifuged and filtered, enhanced the total PCR efficiency up to 70 % while the presence of NH2-MWCNT only centrifuged after sonication, inhibited the reaction significantly at similar concentrations. Furthermore, the NH2-MWCNTs coupled covalently onto magnetic microspheres, contributed for the specificity enhancement whilst decreasing the amplification efficiency by 30 % at the maximum concentration, which suggests a removable enhancement system for sensitive applications. On the other hand, the relative hydrodynamic size distribution measurements displayed a clear difference between the filtered NH2 and non-filtered NH2-MWCNT water dispersions, which justifies the inhibition of the amplification by the non-filtered NH2-MWCNTs containing big agglomerates and bundles. Finally, we demonstrated that major PCR components adsorb onto the NH2-MWCNTs with diverse affinities, and maintain their functions after adsorption, which provides a good framework to further develop tunable NH2-MWCNT-carriers to be utilized in various nanobiotechnology and material science applications

Trends in aptamer selection methods and applications

Aptamers are target specific ssDNA, RNA or peptide sequences generated by an in vitro selection and amplification method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment), which involves repetitive cycles of binding, recovery and amplification steps. Aptamers have the ability to bind with a variety of targets such as drugs, proteins, heavy metals, and pathogens with high specificity and selectivity. Aptamers are similar to monoclonal antibodies regarding their binding affinities, but they offer a number of advantages over the existing antibody-based detection methods, which make the aptamers promising diagnostic and therapeutic tools for future biomedical and analytical applications. The aim of this review article is to provide an overview of the recent advancements in aptamer screening methods along with a concise description of the major application areas of aptamers including biomarker discovery, diagnostics, imaging and nanotechnology

Characterization of a dual biotin tag for improved single stranded dna production

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays. Several methods are available in the literature to produce single-stranded DNA from double-stranded DNA template, such as extraction of the sense strand from denaturing gels, asymmetric PCR, use of streptavidin-biotin interaction, and some alternative methods, including enzymatic digestion of negative strand by either Lambda exonuclease or T7 Gene 6 exonuclease. In this report, a detailed characterization of dual biotin tag method to generate single-stranded DNA from the random oligonucleotide library is presented. Unlike the traditional streptavidin-biotin method that uses single biotin tagged molecule during separation, this novel technique is based on a dual biotin molecule covalently attached to 5’ end of the negative strand. Improved technique takes less than one hour as a consequence of eliminated alkali treatment step, which make this procedure the shortest procedure described in literature so far for single-stranded DNA production. The method can achieve a single-stranded DNA yield around 75% from the corresponding DNA template in Tris-HCl buffer. A number of parameters, such as the effect of different elution buffers and heat treatments, spontaneous release of streptavidin from the magnetic bead surface, loss of beads during consecutive washes, aggregation of the beads, were investigated to reveal the optimal conditions for single-stranded DNA production. FTIR, DLS, SEM, and Electrophoresis techniques were used for characterization studies

Amplification yield enhancement of short DNA templates using bulk and surface-attached amine-functionalized single-wall carbon nanotubes

In this report, water dispersions of pristine, amine-functionalized, and carboxyl-functionalized single-wall carbon nanotubes were introduced into model DNA amplification reactions in order to exploit the effects of surface charge, dispersion quality, and concentration of the carbon nanomaterials in the final amplification yield. The magnetic beads that were covalently modified with the functionalized carbon nanotubes were also evaluated under equal conditions to observe the difference between bulk and surface-attached nanotubes. The centrifugation and filtration steps applied to the bulk dispersions were found to be useful to eliminate aggregates, which ultimately enhanced the final amplification yields of the samples containing pristine and amine-functionalized carbon nanotubes. However, the carboxylated carbon nanotubes displayed an inhibitory action in all samples, regardless of the centrifugation and filtration, indicating a disfavored surface charge. The magnetic beads modified with the amine-functionalized carbon nanotubes also improved the amplification yield; besides, they greatly simplified the elimination of the nanotubes from reactions. Mechanism studies proved the preferential binding of the DNA templates onto the amine-functionalized nanotubes during amplification. Meanwhile, none of the DNA templates showed interaction with the carboxyl-functionalized carbon nanotubes, probably due to the electrostatic repulsion between the negative charges