Use of Ultrastructuke and DNA Hybridization to Detect Replication of the Nuclear Polyhedrosis Virus in Midgut Cells of Homologous Host

It was controversial issue if nuclear polyhedrosis virus(NPV) could replicate in midgut cells of host larvae from Lepidoptera by now.The replication of Mythimna separata NPV(MsNPV) in M.separata larvae midgut cells was studied by ultrastructural and DNA hybridized techniques.The paper demonstrated that the MsNPV could neither infect midgut cell nor replicate in midgut cell of homologous host.Therefore MsNPV virions released from the virial occlusion bodies were considered as direct penetration though the intercellular spaces of midgut cells to hemocoel of the host larvae.

Detection of Breast Cancer 1 (BRCA1) Gene Using an Electrochemical DNA Biosensor Based on Immobilized ZnO Nanowires

Herein we report an electrochemical DNA biosensor for the rapid detection of sequence (5’ AAT GGA TTT ATC TGC TCT TCG 3’) specific for the breast cancer 1 (BRCA1) gene. The proposed electrochemical genosensor is based on short oligonucleotide DNA probe immobilized onto zinc oxide nanowires (ZnONWs) chemically synthesized onto gold electrode via hydrothermal technique. The morphology studies of the ZnONWs, performed by field emission scanning electron microscopy (FESEM), showed that the ZnO nanowires are uniform, highly dense and oriented perpendicularly to the substrate. Recognition event between the DNA probe and the target was investigated by differential pulse voltammetry (DPV) in 0.1 M acetate buffer solution (ABS), pH 7.00;as a result of the hybridization, an oxidation signal was observed at +0.8 V. The influences of pH, target concentration, and non-complimentary DNA on biosensor performance were examined. The proposed DNA biosensor has the ability to detect the target sequence in the range of concentration between 10.0 and 100.0 μM with a detection limit of 3.32 μM. The experimental results demonstrated that the prepared ZnONWs/Au electrodes are suitable platform for the immobilization of DNA.

Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

Microcontact Printing Techniques in Bioscience

The microcontact printing(μCP) technology available for patterning protein, DNA hybridization, immunoassay and cellular cocultures onto solid surface are reviewed. This review describes some of the techniques currently employed for creating two-dimensional biomolecular microarray, and the research results regarding their effectiveness. In addition, the applications of the impact of μCP technology in the field of biosciences are also presented.

The kinetics of force-dependent hybridization and strand-peeling of short DNA fragments

Deoxyribonucleic acid(DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded(ss) strands, forming a double-stranded(ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.

ROLE OF COLPOSCOPY IN THE DIAGNOSIS OF HUMAN PAPI- LLOMAVIRUS INFECTION OF THE UTERINE CERVIX

Among 6706 women screened by cytology, only 9 (0.13%) showed evidence of human pppillomavirus infection (HPVI). In 133 women examined by colposcopy for abnormal cytology or/ and suspected lestions on the cervix, 41.(30. 8%) showed subclinical papillomavirus infection (SPI), while 17. 4% and 5. 3% showed HPVI by histopathology and cytology, respectively. The conformation rate between colposcopy and pathology was 69. 6%. Sixty-nine specimens out of 133 colposcopy piled biopaies were assayed by HPV-DNA dot hybridization with 6B/11, 16, 18 probes to detect the presence of HPV-DNA In the cervical specimens. Thirty-nine (56.5%) gave a positive result. The colposcopic predictive value of positive result for HPVI was 76.7%. The difference between colposcopy (59%) and pathology (20. 5%) is statistically significant (P<0. 01). These results suggest that colposcopy is superior to cytology and hjstopathology for the detection of SPI in the cervix. In colposcopy HPV-DNA positive women, aceto while epithelium was most common (28. 2%) . As it is difficult to differentiate SPI from cervical intraepithelial neoplasia especially the Grade Ⅰ lesion by colposcopy, discrimination criteria are proposed together with the chief colposcopic features of SPI.

Preparation of Digoxigenin labelled Probe and Detection of HBV DNA in Liver and Extrabepatic Tissue with in Situ Hybridization

A new rapid technique for intrahepatic and extrahepatic HBV DNA detection by using digoxigenin labelled probe with in situ hybridization was developed.This technique has the advantage of being non-radioactive and a quick procedure yielding stable results and showing a clear background.

Direct Electrochemical Detection of Oligonucleotide Hybridization on Poly（thionine） Film

In this work, the application of a conducting polymer, poly（thionine）, modified electrode as matrix to DNA immobilization as well as transducer to label-free DNA hybridization detection was introduced. The electropolymerization of thionine onto electrode surface was carried out by a simple two-step method, which involved a preanodization of glassy carbon electrode at a constant positive potential in thionine solution following cyclic voltammetry scans in the solution. Electrochemical detection was performed by differential pulse voltammetry in the electroactivity potential domain of poly（thionine）. The resulting poly（thionine） modified electrode showed a good stability and electroactivity in aqueous media during a near neutral pH range. Additionally, the pendant amino groups on the poly（thionine） chains enabled poly（thionine） modified electrode to immobilize phosphate group terminated DNA probe via covalent linkage. Hybridization process induced a clear decrease in poly（thionine） redox current, which was corresponding to the decrease in poly（thionine） electroactivity after double stranded DNA was formed on the polymer film. The detection limit of this electrochemical DNA hybridization sensor was 1.0 × 10^-10mol/L. Compared with complementary sequence, the hybridization signal values of 1-base mismatched and 3-base mismatched samples were 63.9% and 9.2%, respectively.

Fabrication and characterization of gold nano particles for DNA biosensor applications

This research involves the preparation ofa biosensor using silicon oxide for biomedical applications, and its effective use for the detection of target DNA hybridization. An electrochemical DNA biosensor was successfully fabricated by using （3-aminopropyl） tri-ethoxysilane （APTES） as a linker molecule combined with gold nanoparticles （GNPs） on a thermally oxidized 5i02 thin film. The size of the GNPs was calculated by utilizing UV-vis data with an average calculated particle size within the range of 30 - 5 nm, and characterization by transmission electron microscopy （TEM） and atomic force microscopy （AFM）. The GNP-modified SiO2 thin films were electrically characterized through the measurement of capacitance, permittivity and conductivity using a low-cost dielectric analyzer. The capacitance, permittivity and conductivity profiles of the fabricated sensor clearly differentiated DNA immobilization and hybridization.

Enhanced destabilization of mismatched DNA using gold nanoparticles offers specificity without compromising sensitivity for nucleic acid analyses

Here, we report a method that uses gold nanoparticles （AuNPs） to enhance the specificity of DNA hybridization without reducing its detection sensitivity. The conventional stringent wash method utilizes high-temperatureflow-salt conditions to enhance the specificity of DNA hybridization-based assays. This method creates a destabilizing environment for base pairing that affects specific and nonspecific duplexes. Therefore, specificity is achieved at the expense of signal intensity or sensitivity. However, in the proposed wash method, AuNPs predominantly destabilize nonspecific duplexes, offering specificity without compromising sensitivity. This AuNP wash technique has proven to be effective in detecting single nucleotide polymorphisms （SNPs） in genomic samples even at room temperature in a CD-like NanoBioArray （CD-NBA） chip. This method is also robust with sequence variation and is compatible with multiplex DNA analyses on microarrays. Thus, the AuNP wash method could potentially be useful for improving the accuracy of DNA hybridization results.

SURFACE STRESS PROPERTIES OF DNA-MICROCANTILEVER SYSTEMS

For the first time, the connection between surface stress and nanoscopic interac- tions of DNA adsorbed on microcantilever is established by combining Strey＇s mesoscopic liquid crystal theory and Stoney＇s formula. It is shown that surface stress depends not only on biomolec- ular interactions of DNA biofilm but also on mechanical properties of cantilever. Considering the correlativity between grafting density and chain length of DNA chain, we discuss the differences between DNA-microcantilever system and DNA solution system. The major theoretical achieve- ment of this model is to identify the main contributions to surface stress under different detection conditions. This provides guidelines for designing new biosensors with high sensitivity and improved reliability.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability.The telomeric repeat-containing RNA(TERRA)that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop.In tumor cells,R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres(ALT)pathway.Dysregulated R-loops can cause stalled replication forks and telomere instability.However,how R-loops are recognized and regulated,particularly at telomeres,is not well understood.We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination.Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses.In addition,ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway.Using the proximity-dependent biotin identification(BioID)technology,we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases,including DHX9.Importantly,ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9.Our findings suggest that ILF3 may function as a reader of telomeric R-loops,helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

Amplified DNA Detection Sensitivity Using Streptavidin-Biotinylated Protein Complex: Characterization by Electrochemical Impedance Spectroscopy

Thiol terminated oligonucleotide was immobilized to gold surface by self assembly method. A novel amplification strategy was introduced for improving the sensitivity of DNA hybridization using biotin labeled protein streptavidin network complex. This complex can be formed in a cross linking network of molecules so that the amplification of the response signal will be realized due to the big molecular size of the complex. It could be proved from the impedance technique that this amplification strategy caused dramatic improvement of the detection sensitivity. These results give significant advances in the generality and sensitivity as it is applied to biosensing.

DNA and RNA sensor

This review summarizes recent advances in DNA sensor.Major areas of DNA sensor covered in this review include immobilization methods of DNA,general techniques of DNA detection and application of nanoparticles in DNA sensor.

Functional DNA Structures and Their Biomedical Applications

Since the discovery of the double-helix structure in 1953,nucleic acids have been developed from natural genetic codes into functional building blocks in a wide range of biotechnology and materials sciences.Taking advantage of their design diversity and biocompatibility,functional nucleic acids facilitate the“bottom-up”fabrication of nanomaterials that are highly potential for molecular medicine to treat different diseases,such as cancers.The present perspective article introduces recent advances in the use of these unique properties of nucleic acid biopolymers for biomedical applications.Specifically,nanomaterial/nucleic acid hybrid structures for sensing,controlled drug release,programmable intracellular imaging,and apoptosis,as well as logic calculation,are discussed.Furthermore,the detailed operation for both extracellular and intracellular bioactivity regulation with these new design functional nucleic acid nanostructures are fully illustrated.

Recent progress on DNA block copolymer

DNA has gained great attention because of its unique structure,excellent molecular recognition property,and biological functions.When married with versatile synthetic polymers,the DNA conjugated polymer hybrids,known as DNA block copolymers（DBCs）,have been launched and well developed for the syntheses of new materials and nanostructures with different functions in the past several decades.Compared to conventional synthetic block copolymers,using DNA as a building block provides several advantages over other polymer candidates,such as molecular recognition,programmable self-assembly,biocompatibility,and sequence-encoded information.In this review,recent developments in this area will be summarized and meaningful breakthroughs will be highlighted.We will discuss representative examples of recent progress in the syntheses,structure manipulations,and applications of DBCs.

The effect of secondary structures on the generation of characteristic events during the translocation of DNA hybrid through ?-hemolysin

Nanopore has been developed to be a powerful,single-molecule analytical tool for sensing ions,small organic molecules and biomacromolecules such as proteins and DNAs.Generally,the identity of the analyte can be revealed by current amplitude changes and mean dwell time of the analyte binding events.In some cases,generation of highly characteristic current events affords an alternative way of analyte determination with high confidence level.However,we found that secondary structures in DNA/RNA hybrids might severely hinder the generation of signature events during their translocation through?-hemolysin nanopore.In this report,we propose a strategy to add a certain concentration of urea in the buffer solution for single channel recordings and validate that low concentration of urea can effectively denature the secondary structures in DNA hybrids and recover the generation of signature events.This finding might be useful in other secondary structure-related nanopore sensing activities.

Hybridization chain reaction triggered controllable one-dimensional assembly of gold nanoparticles

Assembling and ordering nanomaterials into desirable patterns are considerably significant,since the properties of nanomaterials depend not only on the size and shape,but also on the spatial arrangement among the collective building blocks.In this work,the DNA self-assembly technology of hybridization chain reaction(HCR) provided a convenient method to yield long double-strand DNA(dsDNA) to install gold nanoparticles(AuNPs) into one dimensional assembly along the skeleton of dsDNA.Interestingly,the tunable length of AuNPs assemblies along dsDNA chain could be achieved by adjusting the reaction time of HCR,which is based on the formation of covalent bond between Au and the-SH group of DNA.Compared with weak light scattering of single AuNP,these AuNPs assemblies could be clearly imaged under the dark field microscopy,indicating that the light scattering was greatly improved after assembling.

Graphene-Coated Optical Fiber SPR Biosensor for BRCA1 and BRCA2 Breast Cancer Biomarker Detection:a Numerical Design-Based Analysis

This paper provides a simple hybrid design and numerical analysis of the graphene-coated fiber-optic surface plasmon resonance(SPR)biosensor for breast cancer gene-1 early onset(BRCA1)and breast cancer gene-2 early onset(BRCA2)genetic breast cancer detection.Two specific mutations named 916delTT and 6174delT in the BRCA1 and BRCA2 are selected for numerical detection of breast cancer.This sensor is based on the technique of the attenuated total reflection(ATR)method to detect deoxyribonucleic acid(DNA)hybridization along with individual point mutations in BRCA1 and BRCA2 genes.We have numerically shown that momentous changes present in the SPR angle(minimum:135%more)and surface resonance frequency(SRF)(minimum:136%more)for probe DNA with various concentrations of target DNA corresponding to a mutation of the BRCA1 and BRCA2 genes.The variation of the SPR angle and SRF for mismatched DNA strands is quite negligible,whereas that for complementary DNA strands is considerable,which is essential for proper detection of genetic biomarkers(916delTT and 6174delT)for early breast cancer.At last,the effect of electric field distribution in inserting graphene layer is analyzed incorporating the finite difference time domain(FDTD)technique by using Lumerical FDTD solution commercial software.To the best of our knowledge,this is the first demonstration of such a highly efficient biosensor for detecting BRCA1 and BRCA2 breast cancer.Therefore,the proposed biosensor opens a new window toward the detection of breast cancers.

Linking circular intronic RNA degradation and function in transcription by RNase H1

Circular intronic RNAs(ci RNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from prem RNA splicing, but their turnover and mechanism of action have remained elusive. We report that RNase H1 degrades a subgroup of ci RNAs in human cells. Many ci RNAs contain high GC% and tend to form DNA:RNA hybrids(R-loops) for RNase H1 cleavage, a process that appears to promote Pol II transcriptional elongation at ci RNA-producing loci. One ci RNA, ciankrd52, shows a stronger ability of R-loop formation than that of its cognate pre-m RNA by maintaining a locally open RNA structure in vitro. This allows the release of pre-m RNA from R-loops by ci-ankrd52 replacement and subsequent ci RNA removal via RNase H1 for efficient transcriptional elongation. We propose that such an R-loop dependent ci RNA degradation likely represents a mechanism that on one hand limits ci RNA accumulation by recruiting RNase H1 and on the other hand resolves Rloops for transcriptional elongation at some GC-rich ci RNA-producing loci.