Sequence-Guided Localization of DNA Hybridization Enables Highly Selective and Robust Genotyping.

Genetic variations are always related to human diseases or susceptibility to therapies. Nucleic acid probes that precisely distinguish closely related sequences become an indispensable requisite both in research and clinical applications. Here, a Sequence-guided DNA LOCalization for leaKless DNA detection (SeqLOCK) is introduced as a technique for DNA hybridization, where the intended targets carrying distinct "guiding sequences" act selectively on the probes. In silicon modeling, experimental results reveal considerable agreement (R2  = 0.9228) that SeqLOCK is capable of preserving high discrimination capacity at an extraordinarily wide range of target concentrations. Furthermore, SeqLOCK reveals high robustness to various solution conditions and can be directly adapted to nucleic acid amplification techniques (e.g., polymerase chain reaction) without the need for laborious pre-treatments. Benefiting from the low hybridization leakage of SeqLOCK, three distinct variations with a clinically relevant mutation frequency under the background of genomic DNA can be discriminated simultaneously. This work establishes a reliable nucleic acid hybridization strategy that offers great potential for constructing robust and programmable systems for molecular sensing and computing.

Multi-parameter surface plasmon resonance instrument for multiple nucleic acid quantitative detection.

Multiplex nucleic acid assays can simultaneously detect the characteristics of different target nucleic acids in complex mixtures and are used in disease diagnosis, environmental monitoring, and food safety. However, traditional nucleic acid amplification assays have limitations such as complicated operation, long detection time, unstable fluorescent labeling, and mutual interference of multiplex nucleic acids. We developed a real-time, rapid, and label-free surface plasmon resonance (SPR) instrument for multiplex nucleic acid detection. The multiparametric optical system based on total internal reflection solves the multiplex detection problem by cooperating with linear light source, prism, photodetector, and mechanical transmission system. An adaptive threshold consistency correction algorithm is proposed to solve the problem of inconsistent responsiveness of different detection channels and the inability of quantitative comparison. The instrument achieves label-free and amplification-free rapid detection of these biomarkers for miRNA-21 and miRNA-141, which are widely expressed in breast cancer and prostate cancer. The multiplex nucleic acid detection takes 30 min and the biosensor has good repeatability and specificity. The instrument has a limit of detection (LODs) of 50 nM for target oligonucleotides, and the smallest absolute amount of sample that can be detected is about 4 pmol. It provides a simple and efficient point-of-care testing (POCT) detection platform for small molecules such as DNA and miRNA.

PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs.

Molecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

Indirect surface-enhanced Raman scattering assay of insulin-like growth factor 2 receptor protein by combining the aptamer modified gold substrate and silver nanoprobes.

An indirect aptamer-based SERS assay for insulin-like growth factor 2 receptor (IGF-IIR) protein was developed. The gold substrate and silver nanoparticles (AgNPs) were employed simultaneously to achieve double enhancement for SERS signals. Firstly, the five commercial SERS substrates including Enspectr, Ocean-Au, Ocean-AG, Ocean-SP and Q-SERS substrates were evaluated using 4-mercaptobenzoic acid (4-MBA). The Q-SERS substrate was selected based on low relative standard deviation (RSD, 8.6%) and high enhancement factor (EF, 8.7*105), using a 785 nm laser. The aptamer for IGF-IIR protein was designed to include two sequences: one grafted on gold substrate to specifically capture the IGF-IIR protein and a second one forming a 3' sticky bridge to capture SERS nanotags. The SERS nanotag was composed by AgNPs (20 nm), 4-MBA and DNA probes that can hybridize with the aptamer. Due to the steric-hindrance effect, when the aptamer doesn't combine with IGF-IIR protein, it only can capture the SERS nanotags. Therefore, there was a negative correlation between the concentration of IGF-IIR protein and the intensity of 4-MBA at 1076 cm-1. The detection limit reached to 141.2 fM and linear range was from 10 pM to 1 µM. The SERS aptasensor also exhibits a high reproducibility with an average RSD of 4.5%. The interference test was conducted with other four proteins to verify the accuracy of measuring. The study provides an approach to quantitative determination of proteins based on specific recognition and nucleic acid hybridization of aptamers, to establish sandwich structure for SERS enhancement. Graphical abstractSchematic representation of surface-enhanced Raman scattering (SERS) assay on insulin-like growth factor 2 receptor (IGF-IIR) protein by combining the aptamer modified gold substrate and 4-mercaptobenzoic acid (4-MBA) and DNA probe modified silver nanoparticles.

Primary versus post-treatment apical periodontitis: microbial composition, lipopolysaccharides and lipoteichoic acid levels, signs and symptoms.

OBJECTIVES: To compare the microbial load and composition and to determine the lipopolysaccharides (LPS) and lipoteichoic acid (LTA) concentrations found in primary apical periodontitis (PAP) and post-treatment apical periodontitis (PTAP), correlating these findings with clinical/tomographic features. MATERIAL AND METHODS: Sixty patients with PAP (31) and PTAP (29) were submitted to clinical and tomographic assessment. Samples were collected from each root canal using paper points for microbiological assessment (culture technique and Checkerboard DNA-DNA hybridization) and determination of LPS and LTA levels (limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively). Data were correlated with clinical/tomographic findings and statistically analyzed using the Mann-Whitney and Pearson correlation tests (α = 5%). RESULTS: A higher number of cultivable bacteria and LPS were found in PAP (p < 0.05). The median number of species per root canal found in PAP and PTAP was 9 and 22, respectively (p < 0.05). LPS was positively correlated with a larger periapical lesion volume (p < .05). LTA levels were similar in both infections and had no correlation with signs and symptoms. In PAP, gram-positive bacteria were correlated with spontaneous pain (p < .05) and exudate (p < .05). Tenderness to percussion and pain on palpation were correlated to the presence of both gram-positive and negative bacteria. In PTAP, a positive correlation was observed between both gram-positive and gram-negative bacteria with exudate and periapical lesion volume (p < .05). CONCLUSIONS: PAP had higher contents of microbial load and LPS compared with PTAP. However, PTAP presented a more diverse microbiota compared with PAP. Higher content of LPS was positively correlated with larger periapical bone destruction, whereas signs and symptoms with specific microorganisms. CLINICAL RELEVANCE: It was verified that PAP and PTAP are polymicrobial infections with predominance of gram-negative bacteria and a more diverse bacterial population found in PTAP. A wide interaction of specific microbial species resulted in different clinical features in both infections.

Biomedical applications of nanoflares: Targeted intracellular fluorescence probes.

Nanoflares are intracellular probes consisting of oligonucleotides immobilized on various nanoparticles that can recognize intracellular nucleic acids or other analytes, thus releasing a fluorescent reporter dye. Single-stranded DNA (ssDNA) complementary to mRNA for a target gene is constructed containing a 3'-thiol for binding to gold nanoparticles. The ssDNA "recognition sequence" is prehybridized to a shorter DNA complement containing a fluorescent dye that is quenched. The functionalized gold nanoparticles are easily taken up into cells. When the ssDNA recognizes its complementary target, the fluorescent dye is released inside the cells. Different intracellular targets can be detected by nanoflares, such as mRNAs coding for genes over-expressed in cancer (epithelial-mesenchymal transition, oncogenes, thymidine kinase, telomerase, etc.), intracellular levels of ATP, pH values and inorganic ions can also be measured. Advantages include high transfection efficiency, enzymatic stability, good optical properties, biocompatibility, high selectivity and specificity. Multiplexed assays and FRET-based systems have been designed.

A ratiometric electrochemical aptasensor for ultrasensitive determination of adenosine triphosphate via a triple-helix molecular switch.

A ratiometric electrochemical aptamer-based assay is described for the ultrasensitive and highly specific determination of adenosine triphosphate (ATP). It is based on ATP aptamer-mediated triple-helix molecular switch (THMS). The method uses (a) a hairpin DNA (MB-DNA-SH) labeled with the redox probe Methylene Blue (MB) at the 3' end, and a thiol group at the 5' end, and (b) a single strand ATP aptamer modified with two ferrocenes at each end (Fc-DNA-Fc). The labeled probe of type MB-DNA-SH was self-assembled onto the surface of a gold electrode via gold-thiol binding. On exposure to Fc-DNA-Fc, it will hybridize with MB-DNA-SH to form a stable THMS structure on electrode surface. In the presence of ATP, it hybridizes with the loop portion of Fc-DNA-Fc, and this results in the unwinding of the THMS structure. Such variation caused the changes of the differential pulse voltammetry (DPV) peak currents of both MB (at around -0.25 V) and Fc (at around 0.39 V; both vs. Ag/AgCl). A significant enhancement is found for the ratio of the two DPV peaks. Under the optimum experimental conditions, this assay has a response that covers the 0.05 to 100 pM ATP concentration range, and the detection limit is 5.2 fM (for S/N = 3). The method is highly selective for ATP over its analogs. Graphical abstract Schematic presentation of a novel ratiometric electrochemical aptasensor for ATP via triple-helix molecular switch (THMS) strategy. MB-DNA-SH was self-assembled on GE surface through gold-thiol binding. Fc-DNA-Fc hybridized with MB-DNA-SH to form THMS structure. ATP specifically bond with its aptamer sequence of Fc-DNA-Fc to unwind the THMS structure. The ratio of DPV peak currents of MB and Fc was applied to monitor the concentration of ATP in real samples over its analogs.

Toehold integrated molecular beacon system for a versatile non-enzymatic application.

A molecular beacon (MB) is an oligonucleotide hybridization probe with a hairpin-shaped structure that leads to specific and instantaneous nucleic acid hybridization, enabling a variety of applications. However, integration of additional module sequences interferes with the performance of MBs and increases the complexity of sequence design. Herein, we develop and characterize a toehold integrated molecular beacon (ToMB) strategy for nucleic acid hybridization, where the reaction rate can be flexibly regulated by a target-induced MB conformational switch. Using this basic mechanism, the ToMB is capable of identifying nucleic acids with high specificity and a wider linearity range compared with the conventional molecular beacon system. We further applied the ToMB to the construction of a hybridization chain reaction system and a basic OR logic gate VJHto explore its programmability and versatility. Our results strongly suggest that the novel ToMB can act as a powerful nano-module to construct universal and multifunctional biosensors or molecular computations. Graphical abstract Molecular beacon is employed as a flexible and switchable spacer to control the toehold-mediated strand displacement reaction.

FastPCR: An in silico tool for fast primer and probe design and advanced sequence analysis.

Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html. Accession numbers for the sequences resulting from this study: EU140956 EU177767 EU867815 EU882730 FJ975775-FJ975780 HM481419 HM481420 KC686837-KC686839 KM262797.

An Impedimetric Biosensor Based on Ionic Liquid-Modified Graphite Electrodes Developed for microRNA-34a Detection.

In the present work, an impedimetric nucleic acid biosensor has been designed for the purpose of detection of microRNA (miRNA). Ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL))-modified chemically activated pencil graphite electrodes (PGEs) were used for the sensitive and selective detection of miRNA-34a. After covalent activation of the PGE surface using covalent agents (CAs), the ionic liquid (IL) was immobilized onto the surface of the chemically activated PGE by passive adsorption. The electrochemical and microscopic characterization of the IL/CA/PGEs was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM). DNA probe concentration, miRNA target concentration, and also the hybridization time and wet adsorption time were optimized by using the EIS technique. Then, the hybridization occurred between specific DNA probes and miRNA-34a was immobilized onto the surface of the IL/CA/PGEs. The impedimetric detection of miRNA-DNA hybrid was performed by EIS. The detection limit (DL) was calculated in a linear concentration range of 2⁻10 µg/mL miRNA-34a target, and it was found to be 0.772 µg/mL (109 nM) in phosphate buffer solution (PBS) and 0.826 µg/mL (117 nM) in diluted fetal bovine serum (FBS). The selectivity of impedimetric biosensor for miRNA-34a was also tested against to other non-complementary miRNA sequences both in buffer media, or diluted FBS.

Synergistic Combination of Unquenching and Plasmonic Fluorescence Enhancement in Fluorogenic Nucleic Acid Hybridization Probes.

Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes.

Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling.

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence-labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine-containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80-fold and high brightness up to 50 mL mol-1 cm-1 ). The detection system provides sub-nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20-fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH-based readout of the bivalent CysCys-PNA display was interfaced with a rolling-circle amplification (RCA) assay used to detect disease-associated microRNA let-7a.

Clamped Hybridization Chain Reactions for the Self-Assembly of Patterned DNA Hydrogels.

DNA hydrogels hold great potential for biological and biomedical applications owing to their programmable nature and macroscopic sizes. However, most previous studies involve spontaneous and homogenous gelation procedures in solution, which often lack precise control. A clamped hybridization chain reaction (C-HCR)-based strategy has been developed to guide DNA self-assembly to form macroscopic hydrogels. Analogous to catalysts in chemical synthesis or seeds in crystal growth, we introduced DNA initiators to induce the gelation process, including crosslinked self-assembly and clamped hybridization in three dimensions with spatial and temporal control. The formed hydrogels show superior mechanical properties. The use of printed, surface-confined DNA initiators was also demonstrated for fabricating 2D hydrogel patterns without relying on external confinements. This simple method can be used to construct DNA hydrogels with defined geometry, composition, and order for various bioapplications.

Comparison of MALDI-TOF MS, nucleic acid hybridization and the MPT64 immunochromatographic test for the identification of M. tuberculosis and non-tuberculosis Mycobacterium species.

Mycobacteria are an important cause of morbidity in humans. Rapid and accurate mycobacterial identification is important for improving patient outcomes. However, identification of Mycobacterium species is not easy, due to the slow and fastidious growth of mycobacteria. Recently, biochemical, sequencing, and probing methods have come to be used for identification. This study compared the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of M.tuberculosis and non-tuberculosis Mycobacteria (NTM) to those of nucleic acid hybridization (NAH) and the MPT64 immunochromatographic test. A total of 69 isolates from Marmara University Hospital, Microbiology Laboratory obtained between 2012 and 2013 were included in our study. All strains were grown on Lowenstein-Jensen and Middlebrook 7H9 medium. Among the 69 isolates, 56 (81%) were isolated as Mycobacterium tuberculosis complex (MTC), and 13 (19%) were isolated as NTM by the MPT64 ICT. NAH was able to identify all isolates to the species level. The isolated NTM included M. intracellulare (n:5), M. lentiflavum (n:3), M. xenopi (n:2), M. malmoense (n:1), M. abscessus (n:1), and M. avium (n:1). MALDI-TOF MS identified 88% of the mycobacterial isolates. All M. tuberculosis strains were identified correctly, but the ratio was 38.5% for NTM. Mycobacterial identification using MALDI-TOF MS takes 45 minutes and costs 3 Euro/test, whereas mycobacterial identification using NAH takes 6-7 hours and costs 30 Euro/test. In conclusion, MALDI-TOF MS has the potential to identify mycobacteria in the clinical laboratory setting by reducing identification turnaround time and laboratory costs for isolate referral.

Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids.

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations.

Performance of REBA MTB-XDR to detect extensively drug-resistant tuberculosis in an intermediate-burden country.

Extensively drug-resistant tuberculosis (XDR-TB) is a serious worldwide problem. The REBA MTB-XDR (REBA-XDR) was recently developed in Korea to detect resistance to ofloxacin, kanamycin, and streptomycin. The aim of this study is to evaluate the diagnostic accuracy of the REBA-XDR. We prospectively enrolled 104 patients with acid-fast bacilli smear-positive specimens between July 2010 and January 2013. Performance characteristics were compared between REBA-XDR and conventional drug-susceptibility testing. The sensitivity values of REBA-XDR for detecting resistance to ofloxacin, kanamycin, and streptomycin were 66.7%, 90.9%, and 60.0%, and the specificity values were 93.3%, 93.5%, and 85.4%, respectively. The positive predictive values were 62.5%, 62.5%, and 40.9%, and the negative predictive values were 94.3%, 98.9%, and 92.7%, respectively. Accuracy was 89.4%, 93.3%, and 81.7%, respectively. REBA-XDR seems to be a useful kit for "ruling in" XDR-TB in intermediate-burden countries, and especially useful for detecting kanamycin resistance.

Comparison between fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis in translocation carriers.

OBJECTIVES: To compare the pregnancy outcome of the fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis of translocation carriers. DESIGN: Historical cohort. SETTING: A teaching hospital in Hong Kong. PATIENTS: All preimplantation genetic diagnosis treatment cycles performed for translocation carriers from 2001 to 2013. RESULTS: Overall, 101 treatment cycles for preimplantation genetic diagnosis in translocation were included: 77 cycles for reciprocal translocation and 24 cycles for Robertsonian translocation. Fluorescent in-situ hybridisation and array comparative genomic hybridisation were used in 78 and 11 cycles, respectively. The ongoing pregnancy rate per initiated cycle after array comparative genomic hybridisation was significantly higher than that after fluorescent in-situ hybridisation in all translocation carriers (36.4% vs 9.0%; P=0.010). The miscarriage rate was comparable with both techniques. The testing method (array comparative genomic hybridisation or fluorescent in-situ hybridisation) was the only significant factor affecting the ongoing pregnancy rate after controlling for the women's age, type of translocation, and clinical information of the preimplantation genetic diagnosis cycles by logistic regression (odds ratio=1.875; P=0.023; 95% confidence interval, 1.090-3.226). CONCLUSION: This local retrospective study confirmed that comparative genomic hybridisation is associated with significantly higher pregnancy rates versus fluorescent in-situ hybridisation in translocation carriers. Array comparative genomic hybridisation should be the technique of choice in preimplantation genetic diagnosis cycles in translocation carriers.

Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL)) modified pencil graphite electrode (IL-PGEs) was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC). The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV) technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC) or mismatched DNA sequence (MM), or and in the presence of mixture of DNA target: NC (1:1) and DNA target: MM (1:1).

Multiplexed programmable release of captured DNA.

Nucleic-acid hybridization is widely used for the specific capture of complementary sequences from complex samples. It is useful for both analytical methodologies, such as array hybridization (e.g. transcriptome analysis, genetic-variation analysis), and preparative strategies such as exome sequencing and sequence-specific proteome capture and analysis (PICh, HyCCAPP). It has not generally been possible to selectively elute particular captured subsequences, however, as the conditions employed for disruption of a duplex can lack the specificity needed to discriminate between different sequences. We show here that it is possible to bind and selectively release multiple sets of sequences by using toehold-mediated DNA branch migration. The strategy is illustrated for simple mixtures of oligonucleotides, for the sequence-specific capture and specific release of crosslinked yeast chromatin, and for the specific release of oligonucleotides hybridized to DNA microarrays.

Strategies for optimizing DNA hybridization on surfaces.

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.