Biotinylated DNA Probes Research Articles

Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples.

The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples (serum, plasma) is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5′ tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5′ tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5′ tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs.

Development and evaluation of enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for diagnosis of invasive aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection in immunocompromised patients, rapid and sensitive detection of Aspergillus from clinical samples has been a major challenge in the early diagnosis of IA. An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed to fulfil the need for the efficient diagnosis of these infections. The primers targeting 18S rRNA were selected for the amplification of Aspergillus RNA by the isothermal digoxigenin (DIG)-labeling NASBA process. The DIG-labeled RNA amplicons were hybridized with a specific biotinylated DNA probe immobilized on streptavidin-coated microtiter plate. The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to ALP and substrate (disodium 4-nitrophenyl phosphate). The detection limit of the Aspergillus NABSA-ELISA system was 1 CFU and the RNA in non-target bacteria or fungus was not amplified. The performance of this NASBA-ELISA compared to RT-PCR and galactomannan (GM) was evaluated by testing blood samples from 86 patients at high risk for IA. The sensitivity of NASBA-ELISA, RT-PCR and GM-ELISA was 80.56 % (95 % CI 63.98–91.81), 72.22 % (95 % CI 54.81–85.80), 58.33 % (95 % CI 40.76–74.49), respectively, and the specificity was 80.00 % (95 % CI 66.28–89.97), 84.00 % (95 % CI 70.89–92.83), 82.00 % (95 % CI 68.56–91.42). The efficiency of the three methods in various combinations was also evaluated. Combination of NASBA-ELISA and GM-ELISA testing achieved perfect specificity (100 %; 95 % CI 92.89–100) and perfect positive predictive value (100 %; 95 % CI 83.16–100). The best sensitivity (97.22 %; 95 % CI 85.47–99.93) and the highest Youden index (0.652) were obtained by testing with both NASBA and RT-PCR in parallel. In conclusion, the NASBA-ELISA assay consists of an alternative process for large-scale samples detection with semi-quantitative results and provides good clinical performance without resorting to expensive equipment. This assay makes it possible for the NASBA based RNA diagnosis to become a routine work in laboratories in less developed countries with fewer resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0266-0) contains supplementary material, which is available to authorized users.

Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.

The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

Abstract 2846: Ultrasensitive detection of actionable subclones in chronic myeloid leukemia

Abstract Targeted Abl inhibitors such as imatinib have vastly improved survival in chronic myeloid leukemia (CML). However resistance occurs in many patients, most frequently due to point mutations in the Abl kinase. At the time of diagnosis, resistance mutations may already exist in a small fraction of the malignant cells. Second-generation inhibitors have been developed which can overcome specific resistance mutations. Identification of pre-existing resistance mutations would thus have important therapeutic implications, as it could inform which therapy would result in the most durable response in a given patient. Prior searches for pre-existing Abl mutations in CML have used methods such as allele-specific PCR or next-generation DNA sequencing; these methods are prone to background errors resulting in frequent false positive mutations. For example, next-generation sequencing has an error rate of 0.1%-1%, which results in a large number of erroneous mutations that obscure true variants. Enrichment of the Abl gene is also a major limitation. Nearly all prior reports have used PCR amplification for dozens of cycles to enrich the Abl gene prior to mutation detection; this process introduces thousands of PCR errors which make differentiation of true mutations from artifactual mutations challenging. We have developed a protocol for DNA sequencing, Duplex Sequencing, that separately tags and sequences each of the two strands of single DNA molecules. True mutations are present at the same position in both strands and are complementary, while PCR or sequencing errors occur in only one of the strands and are not scored. Duplex Sequencing has a calculated error rate of less than one artifactual mutation per billion nucleotides sequenced. To apply Duplex Sequencing to the Abl oncogene, we established a new, extremely efficient targeted capture approach that utilizes sequential rounds of capture with biotinylated DNA probes. This protocol results in &amp;gt;1,000,000 fold enrichment, with up to 99.8% of reads corresponding to the Abl gene. Duplex Sequencing was carried out at high depth (&amp;gt;100,000 fold), resulting in an average of 2,000 duplex reads per nucleotide position. The estimated accuracy for mutation identification was 99.9999%. We are in the process of sequencing samples from CML at the time of diagnosis and relapse to assay for drug resistance mutations below the limit of detection of conventional assays. Our approach could be extended to any targeted molecular agent for which mutation of the target confers resistance to the drug. Citation Format: Michael W. Schmitt, Bella Aminov, Jerald P. Radich, Lawrence A. Loeb. Ultrasensitive detection of actionable subclones in chronic myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2015-2846

Site-specific immunochemical methylation assessment from genome DNA utilizing a conformational difference between looped-out target and stacked-in nontarget methylcytosines.

We report the sequence-selective immunochemical discrimination of methylcytosine from genomic DNA that we achieved by utilizing selective antibody binding to a looped-out methylcytosine in a bulge region and without using bisulfite treatment, a methylation-sensitive restriction enzyme, or PCR. First, we investigated the affinity of an anti-methylcytosine antibody for methylcytosine in full match, bulge, mismatch and abasic sites using surface plasmon resonance measurement and a microtiter plate to explore the differences as regards antibody binding to the target methylcytosine. The highest affinity, which was comparable to that in a single strand of DNA, was observed in the bulge region. In particular, no affinity was observed in a full match site. This is because there is no interaction such as hydrogen bond or π-π stacking for the bulged methylcytosine, thus enabling only the target in the bulge to be looped out. Methylated and unmethylated genomic DNA were blended to form a model DNA with which to assess the methylation ratio at a specific site. Fragmented DNA was hybridized with a biotinylated probe DNA, which has a sequence capable of forming a single base bulge at the target. The probe design is simple because it consists solely of the elimination of guanine paired with the target cytosine from a full match sequence. As a result, we successfully obtained a linear relationship (r(2)=0.9962) between the immunoassay signal and the methylation ratio of a specific site within 4 h.

Detection of DNA Hybridization Using Protein A Modified Ion Sensitive Field Effect Transistor

A DNA sensor based on an ion sensitive field effect transistor (ISFET) with protein A modification for characterization of DNA hybridization is reported. The surface of the ISFET was modified with protein A via physical adsorption. Protein A binds specifically to the crystallizable fragment of the antibody leading to uniform orientation of the antigen binding site. In order to characterize DNA hybridization, the antibiotin antibody was immobilized to protein A and a single-strand biotinylated DNA probe was added to bind to a specific antibiotin antibody. The voltage shift of the DNA hybridization was observed after the complementary DNA target was added to the immobilized probe. The noncomplementary DNA target was also tested as a negative control. The optimal concentrations were 1 mg/mL for protein A, 1 mg/mL for the anti-biotin antibody, 0.5 µM for the biotinylated probe, and 0.5 µM for the synthetic DNA target. Nevertheless, high concentrations of protein A and antibiotin may prevent the hybridization signal detection; therefore, 0.1 mg/mL of protein A and antibiotin were employed instead of their optimal concentrations. By this strategy, DNA hybridization of the synthetic target was successfully measured with a limit of detection of approximately 0.08 µM. In addition, complementary DNA was differentiated from a noncomplementary DNA target.

Improved solid-phase DNA probe method for tRNA purification: large-scale preparation and alteration of DNA fixation.

The solid-phase DNA probe method, in which a target transfer RNA (tRNA) is hybridized with a complementary DNA oligomer, is generally used for tRNA purification. However, purification of tRNAs from thermophiles by this method is not easy because of their high melting temperatures. To overcome this problem, the use of tetraalkylammonium salts was previously reported [Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S., and Nishikawa, K. (2010) Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38, e89]. In this study, we initially devised a large-scale purification system using tetraalkylammonium salts. The yield of tRNA was increased more than 10-fold and the manual steps were decreased as compared with the previous procedure. However, deterioration of column was very rapid owing to shedding of the biotinylated DNA probe. We therefore devised a method of covalent DNA fixation, in which a 5'-aminohexyl (dT)8 oligomer was fixed onto the N-hydroxysuccinimide-activated agarose, and then a DNA oligomer containing the tRNA and repeated A8 sequences was annealed. The probe sequence for tRNA purification was synthesized in column with Klenow enzyme. This DNA fixation method enabled us to use the column repeatedly and to wash the column with warmed buffers. Thus, this DNA fixation method is economical as compared with the previous method using the biotinylated DNA probe.

Determination of Lead(II) by a Nitrocellulose Membrane Fluorescent Biosensor Based on G-Quadruplex Conformational Changes

A simple, label-free fluorescence method was developed for the sensitive determination of lead(II) using a nitrocellulose membrane biosensor. The surface of the nitrocellulose membrane was modified by glutaraldehyde to conjugate streptavidin, followed by the immobilization of a DNA probe via a biotin modifier. The biotinylated DNA probe can fold into a G-quadruplex structure in the presence of potassium ion that selectively binds to N-methyl mesoporphyrin IX and yields a strong fluorescence signal. The presence of lead(II) can induce a conformational change of the G-quadruplex to a more compact structure, which results in the release of potassium ion and N-methyl mesoporphyrin IX with a concomitant reduction of the fluorescence signal. The biosensor displayed a detection limit as low as 10 nM with excellent selectivity for lead(II) over other metal ions. The developed biosensor was employed for the determination of lead(II) in spiked river water.

Visible-light induced photoelectrochemical biosensor for the detection of microRNA based on Bi2S3 nanorods and streptavidin on an ITO electrode

We demonstrate a photo-electrochemical biosensor for the sensitive and specific detection of microRNA using Bi2S3 nanorods as a photoactive material and streptavidin as the unit that inhibits photocurrent. Bi2S3 nanorods were synthesized hydrothermally in organic phase and displayed excellent light-to-current conversion efficiency. The Bi2S3 was deposited on an indium tin oxide (ITO) slice and then modified with gold nanoparticles onto which biotinylated hairpin probe DNA was deposited as a monolayer. Following hybridization between the biotinylated probe DNA and the target microRNA, the stem-loop structure of the probe DNA was unfolded and the biotin directed outwards into the solution. Streptavidin was then added to bind to biotin via the strong streptavidin-biotin interactions. This causes the photocurrent of the modified ITO to decrease due to steric hindrance that blocks the transfer of electrons from added ascorbic acid to the surface of the electrode. The method has a detection limit as low as 3.5 fM of microRNA and can excellently discriminate even singly mismatched microRNA. The method was successfully applied to investigate the effect of abscisic acid on the expression level of microRNA-159a in seeds of Arabidopsis thaliana. We conclude that the assay presented here has a large potential as a method for quantification of microRNA and for studying the epigenetic regulation of flowering plants.

An impedimetric study of DNA hybridization on paper-supported inkjet-printed gold electrodes

In this study, two different supramolecular recognition architectures for impedimetric detection of DNA hybridization have been formed on disposable paper-supported inkjet-printed gold electrodes. The gold electrodes were fabricated using a gold nanoparticle based ink. The first recognition architecture consists of subsequent layers of biotinylated self-assembly monolayer (SAM), streptavidin and biotinylated DNA probe. The other recognition architecture is constructed by immobilization of thiol-functionalized DNA probe (HS-DNA) and subsequent backfill with 11-mercapto-1-undecanol (MUOH) SAM. The binding capacity and selectivity of the recognition architectures were examined by surface plasmon resonance (SPR) measurements. SPR results showed that the HS-DNA/MUOH system had a higher binding capacity for the complementary DNA target. Electrochemical impedance spectroscopy (EIS) measurements showed that the hybridization can be detected with impedimetric spectroscopy in picomol range for both systems. EIS signal indicated a good selectivity for both recognition architectures, whereas SPR showed very high unspecific binding for the HS-DNA/MUOH system. The factors affecting the impedance signal were interpreted in terms of the complexity of the supramolecular architecture. The more complex architecture acts as a less ideal capacitive sensor and the impedance signal is dominated by the resistive elements.

LacR Mutations Are Frequently Observed in Streptococcus intermedius and Are Responsible for Increased Intermedilysin Production and Virulence

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

OP233: Prevalence of P16INK4 expression and correlation with histopathology in squamous cell carcinoma of head and neck [SCCHN

Introduction Epidemiologic and molecular studies have identified high risk type human papilloma viruses (HPV) as a risk factor in Oropharyngeal SCC in the developed countries. However, prevalence of HPV varies widely in literature and ranges from 20 to 75%. P16 immunohistochemistry (IHC) has emerged as a reliable surrogate marker for HPV related oropharyngeal SCC. Purpose 1. Detection of prevalence of HPV in OSCC and oropharyngeal SCC in the Indian patients. 2. Correlation of prevalence of HPV with histologic subtypes of SCC. Material and Methods HE sections of biopsy of SCC of oral cavity and oropharynx were studied. SCC were typed (conventional or variants), further subtyped as keratinising or non-keratinising type and graded as per WHO classification. The antibody to p16INK4[Biogenex] was standardized. Immunostaining was performed by polymer detection technique. Nuclear positivity with or without cytoplasmic positivity in more than 5% of tumour cells considered as positive result. Every 10th case of p16 positive and p16 negative was validated with In situ hybridization [ISH] for high risk HPV. ISH was carried out by using HPV biotinylated DNA probe. Hybridized probe was detected using Zytovision Detection System. Results 66 cases of OSCC and 91 cases of oropharyngeal SCC were analysed. 6% of OSCC and 25% of oropharyngeal SCC were positive for p16. All the p16 positive OSCC and 35% of p16 positive oropharyngeal SCC were kertanising type. 65% of oropharyngeal SCC were poorly differentiated and nonkeratinising type. Tonsil was the most common site of p16 positive oropharyngeal SCC [57%]. Conclusions 1. The overall prevalence of HPV is lower in SCCHN in the Indian patients. 2. The prevalence is higher in the oropharynx than oral cavity. The difference is statistically significant. 3. P16 expression is correlated well with poor differentiation and non-keratinising type histology in the oropharyngeal SCC.

Genomagnetic assay for electrochemical detection of osteogenic differentiation in mesenchymal stem cells

The osteogenic differentiation of mesenchymal stem cells (MSCs) was assessed by determining the gene expression levels of proteins; osteocalcin (OSC), osteonectin (OSN) and osteopontin (OSP) based on electrochemical detection protocol combined with genomagnetic assay in parallel to real-time PCR analysis. Genomagnetic assay was performed using streptavidin coated commercial magnetic particles (magnetic beads, MBs) in combination with single-use electrochemical sensor technology. A biotinylated DNA probe was immobilized onto streptavidin coated magnetic particles, and then the hybridization process of the probe with its complementary DNA was performed. The oxidation signals of DNA electroactive bases guanine and adenine were measured voltammetrically using a pencil graphite electrode (PGE) before and after the hybridization process of OSC/OSN/OSP probe sequences with their complementary target sequences. The selectivity of the genomagnetic assay was also tested using each DNA probe individually related to osteogenic differentiations. The voltammetric detection of osteogenic differentiations was confirmed selectively by real-time PCR analysis.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Abstract 47: Development and Use of a Novel Single-Cell Epigenetic Lineage Tracing Method to Identify Smooth Muscle--Derived Macrophage-Like Cells Within Human Atherosclerotic Lesions

Aim: Smooth muscle cells (SMC) possess remarkable phenotypic plasticity that allows adaptation to changing environmental cues. Lack of definitive SMC lineage tracing studies and inability to identify phenotypically modulated SMCs within lesions due to loss of SMC marker gene expression raise major questions regarding the role of SMC in vivo in atherosclerosis progression. We hypothesize that a subset of cells within lesions that express macrophage markers are derived from SMC not monocytes and play a key role in determining plaque stability. Methods: We developed a novel lineage tracing based on detection of H3K4dime of the SM MHC gene, a SMC-specific epigenetic lineage marker we have previously shown is stable during phenotypic switching in vitro. Detection of H3K4dime of the SM MHC locus was done using a Proximity ligation assay (PLA) developed in our lab with an antibody targeting the biotinylated DNA probe for the SM MHC locus in conjunction with an anti-H3K4dime antibody. Use of secondary antibodies conjugated with oligonucleotides induces formation of circular DNA that serve as template for amplification, allowing visualization of co-localization of H3K4dime and the SM MHC gene (Duolink). Our new lineage tracing is suitable with human paraffin-embedded tissue sections (n=4) allowing investigation of SMC fate within human atherosclerotic lesion. Results: H3K4dime on the SM MHC gene (PLA+ cells) was found to be specific for SMCs and not found in any other cell types including adventitial fibroblasts, or endothelial cells. The method was validated using a SMC-specific lineage tracing mouse model wherein SM MHC Cre mice are crossed to ROSA flox STOP eYFP+/+ ApoE -/- mice. The H3K4dime/SM MHC PLA signal (i.e. PLA+) was exclusively found in eYFP+ cells. Moreover, some of the lesion SMCs were eYFP+/PLA+/SM α-actin-. Similarly, we identified PLA+ cells in human lesions that were positive for the macrophage marker CD68. Conclusion: Our new method permits definitive identification of SMC-derived cells within lesions even if they are not identifiable as SMC due to loss of SMC markers. Moreover, we provide exciting evidence that a significant fraction of macrophage-like cells in human lesions are derived from SMC. We postulate that transition of SMC to a macrophage state may be a key event leading to plaque destabilization and rupture with possible myocardial infarction or stroke.

Electrochemical detection of individual single nucleotide polymorphisms using monobase-modified apoferritin-encapsulated nanoparticles

An electrochemical approach for detection of individual single nucleotide polymorphisms (SNPs) based on nucleobase-conjugated apoferritin probe loaded with metal phosphate nanoparticles is reported. Coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA was induced by DNA polymerase I (Klenow fragment) to preserve Watson–Crick base-pairing rules. After sequential liquid hybridization of biotinylated DNA probes with mutant DNA and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a well known and specific biotin-streptavidin affinity binding. For signaling each of eight possible Single-nucleotide polymorphisms (SNPs), Pb, Cu, Cd and Zn phosphate-loaded apoferritin nanoparticle probes were linked to adenosine (A), cytidine (C), guanosine (G), and thymidine (T) mononucleotides, respectively. Monobase-conjugated apoferritin probes were coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Electrochemical stripping analyses of the metals loaded in apoferritin nanoparticle probes provide a means for detection and quantification of mutant DNA. Each mutation captures different nucleotide-conjugated apoferritin probe and provide a distinct four-potential voltammogram, whose peak potentials reflect the identity of the mismatch. The method is sensitive enough to accurately determine AG mutation, as the most thermodynamically stable mismatch to detect, in the range of 50–600pM. The proposed protocol provides a simple, fast, cost-effective, accurate and sensitive method for detection of SNPs.

Screening Method for Real-Time Detection of Influenza-A Virus in Human Throat Swabs by Surface Plasmon Resonance Biosensor

A surface plasmon resonance (SPR) biosensor was applied to detect influenza-A virus in human. The detection scheme was based on the measurement of SPR response unit resulting from the hybridization of biotinylated DNA probe immobilized on the SPR chip modified with streptavidin and the product of polymerase chain reaction reversed from the influenza-A virus RNA segment (AB514942). The prepared biosensor demonstrated optimum performance in 200 mM phosphate buffered saline (PBS) with a pH value of 7.5 and exhibited good sensitivity with a detection limit of 0.5 pM for perfect complementary hybridization. In addition, the prepared biosensor can effectively discriminate perfect complementary and other three types of mismatch: base substitute, base insertion and base deletion in 200 mM phosphate buffered saline (PBS) with a pH value of 7.5. Furthermore, the influenza-A virus in throat swab samples was directly (without RNA extraction, and amplification) detected with the prepared machine, and the result showed that the SPR response unit was in response to the dilution factor of throat swabs. Better sensitivity and specificity based on Surface Plasmon Resonance biosensor were obtained which demonstrated a promising potentiality in detecting influenza-A virus

Electrochemical DNA biosensor based on avidin–biotin conjugation for influenza virus (type A) detection

An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin–biotin conjugation of a biotinylated probe DNA, 5′-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3′, and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5′-TTC GAC CTC GGT TAG AAG ACT CAT-3′ and two-base mismatched DNA, 5′-TTC GAC AGC GGT TAT AAG ACT CAT-3′.

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.