Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction

The combination of high selectivity of aptamer with the peroxidase-mimicking property of DNAzyme has presented considerable opportunities for designing colorimetric aptasensor for detection of ochratoxin A (OTA). The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. Hybridization chain reaction (HCR) between two hairpin DNAs was employed to further improve the sensitivity of this method. The presence of OTA triggers the opening of the hairpin structure and the beginning of HCR, which results in the release of many DNAzyme, and generates enhanced colorimetric signals, which is correlated to the amounts of OTA with linear range between 0.01 to 0.32 nM, and the limit of detection is 0.01 nM under optimal conditions. OTA in yellow rice wine and wheat flour samples was also detected using this method. We demonstrate that a new colorimetric method for the detection of OTA has been established, which is simple, easy to conduct, label-free, sensitive, high throughput, and cost-saving.     

Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV–vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10⁻¹³ to 4.5 × 10⁻⁶ mol/L. The detection limit (3σ) of this method has decreased to pico-molar level.     

Separation of polymerase chain reaction amplified bird genes by capillary electrophoresis

Female birds possess one Z and one W chromosome, whereas male birds possess two identical Z chromosomes. Thus, the presence of a W genetic marker is diagnostic of the female sex. Capillary electrophoresis with buffer containing an entangled solution of hydroxyethylcellulose was used to separate the polymerase chain reaction (PCR) amplified bird sexing genes CHD-Z and CHD-W. The relative standard deviations (RSD) were less than 0.6% for the male genes and less than 0.4% for the female genes for six runs and detection limits of 0.1 ng/microL were obtained with laser-induced fluorescence (LIF) detection. Using a DNA ladder and theoretical models for DNA separation in sieving media, the sizes of the two bird genes were determined.     

Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence.The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised.The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.     

Label-free and non-enzymatic detection of DNA based on hybridization chain reaction amplification and dsDNA-templated copper nanoparticles

A label-free and non-enzymatic amplification fluorescent method for detection of DNA has been developed by using hybridization chain reaction (HCR) and dsDNA-templated copper nanoparticles (CuNPs). First, the biotinylated capture DNA probes were immobilized on the streptavidin-modified beads through the interaction of biotin and streptavidin. Then, target DNA hybridized with the capture DNA probes, which formed a hybridized DNA with sticky end. The sticky end triggered the HCR process and formation of dsDNA polymers while two hairpin probes coexisted. Subsequently, the dsDNA polymers were employed as template for synthesis of CuNPs with excellent fluorescent properties, which provided a label-free, non-enzymatic signal response. Meanwhile, the fluorescence sensing depended on the target DNA triggered HCR, which render this method a high selectivity against single-base mismatch sequences. The concept and methodology developed in this work show great promise in the quantitative detection of DNA in biological and medical applications.     

A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species

Polymerase chain reation (PCR) fingerprints are used to characterize and recognize bacteria and are generally obtained using universal primers that generate an array of DNA amplicons, which can be separated by electrophoresis. Universal primers 8F and 1491 R have been used to amplify specifically 16S rDNA. We have used these primers at an annealing temperature of 50 degrees C. Agarose gel electrophoresis of PCR products revealed several bands. The band pattern of each bacterial species was different and the strains belonging to the same species shared an identical pattern. The patterns obtained did not show variations with plasmid DNA content or the growth stage of the bacteria. The peculiarity of the randomly amplified polymorphic DNA (RAPD) described in this work lies in the use of two large primers (proximately 20 nt) to obtain the pattern, since normally a only smaller primer is used, and in the new application for the primers used to amplify 16S rDNA. This new procedure, called two primers (TP)-RAPD fingerprinting, is thus rapid, sensitive, reliable, highly reproducible and suitable for experiments with a large number of microorganisms, and can be applied to bacterial taxonomy, ecological studies and for the detection of new bacterial species.     

Real-time polymerase chain reaction detection of fishmeal in feedstuffs

A SYBR Green PCR system was developed for detection of fishmeal in feedstuffs. The real-time PCR method combines the use of fish-specific primers that amplify an 87 base pair (bp) fragment of the mitochondrial 12S ribosomal RNA gene from fish species, and a positive control primer pair that amplifies a 99 bp fragment of the nuclear 18S ribosomal RNA gene in all eukaryotic organisms. The specificity of the primers was tested against 52 animal species and six plant species. Reference feedstuff samples were successfully tested for the presence of fishmeal, demonstrating the applicability of the assay to feedstuffs.     

Direct fluorescence detection of point mutations in human genomic DNA using microbead-based ligase chain reaction

This report has described a convenient genotyping method capable of detecting point mutations directly in human genomic DNA based on the combination of ligase chain reaction (LCR) and microbead-enrichment technique. LCR primers, including a biotin-labeled common primer and two fluorescence-labeled allele-specific primers, are designed for two alleles of a mutated site. When genomic DNA carries the mutated site, the common primer and allele-specific primer are ligated to form exponential amplified biotin-labeled fluorescence ligation products. These ligated products are enriched by streptavidin-coated microbeads, and genotypes are identified conveniently according to the fluorescence color of microbeads using fluorescent microscopy. Due to amplification of LCR process and enrichment of microbeads, the detection limit of the proposed method is as low as 10⁻¹⁵ mol/L templates. The method provides a convenient and simple strategy to detect point mutation directly in human genome. We have confirmed the efficiency of this approach with the identification of β-globin gene point mutation, which results in the reduced production of globin in an inherited hemoglobin disorder thalassemia disease.     

Aptamer-based polymerase chain reaction for ultrasensitive cell detection

A new system was developed for sensitive and selective detection of tumor cells taking advantage of cell-attached aptamers amplified by PCR and output signals amplified by cationic conjugated polymers.     

Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays

A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.     

Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction.

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.     

Label-free and rapid colorimetric detection of DNA damage based on self-assembly of a hemin-graphene nanocomposite

Hemin-graphene nanosheets (H-GNs) can be controllably assembled by target DNA via a hybridization process. This results in a color change from dark blue-green to light blue-green. The degree of aggregation is dependent on DNA concentration and very sensitive to base mismatch. The formation of the blue-green color can be detected with bare eyes or a spectrometer. The method is simple, rapid, and works over the concentration range from 1.0 to 100 nM. The detection limit for target DNA is 0.2 nM. Excellent selectivity is also found in that a DNA with a single base mismatch can be discriminated. This was exploited to detect DNA damage as induced by styrene oxide, sodium arsenite, Fenton’s reagent, or UV radiation. We presume that this method represents a promising tool for evaluating genotoxicity. Figure

Detection of DNA damage based on DNA-directed self-assembly of H-GNs     

Hybridization and polymerase chain reaction amplification of simple repeated DNA sequences for the analysis of forensic stains.

We have evaluated oligonucleotide hybridization and amplification techniques with regard to quantity and quality of genomic DNA that is under investigation in practical forensic case work. In order to obtain sufficient information from analyzing stain material, we use hypervariable simple repeat sequences for individualization, which occur in all eukaryotic genomes. For the analysis of larger amounts of stains (greater than 500 ng DNA) the multilocus probes (CAC)5/(GTG)5* are superior because of their discrimination potential--provided that the hybridizing DNA is of high molecular weight. The less discriminating probes (CT)8 and (GACA)4 are more sensitive (minimal amount: 100ng DNA) and still informative when the DNA is degraded. To increase the sensitivity of forensic stain analysis in special cases we have used the polymerase chain reaction technique to amplify hypervariable simple (gt)n/(ga)m repeat structures from the intron 2 of HLA-DRB genes. Largely independent of the starting amount of DNA and independent of the degradation status, we were able to generate discriminating DNA fragments, which can be used to type (i) microstains and (ii) totally degraded material including human mummy DNA.     

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.     

Conductivity detection of polymerase chain reaction products separated by micro-reversed-phase liquid chromatography

In this paper, we describe the application of micro-reversed-phase high-performance liquid chromatography (mu-RP-HPLC) for the separation and/or purification of polymerase chain reaction (PCR) products with detection accomplished using a miniaturized conductivity detector. The conductivity detector used two Pt wires and a bipolar waveform applied to the electrode pair from which the conductivity of the bulk solution could be measured. In the mobile phase used for the mu-RP-HPLC separation of the PCR product, the mass detection limit for herring sperm DNA using conductivity was found to be 11 ng. Efficient separation of the PCR amplicon from the other reagents present in the PCR cocktail was achieved in less than 4 min with a capacity factor of 2.5 and separation efficiency of 9.1 x 10(3) plates. The separation was carried out using reversed-phase ion-pair chromatography with a triethylammonium acetate ion-pairing agent.     

Quantum dot-based isothermal chain elongation for fluorescence detection of specific DNA sequences via template-dependent surface-hybridization

A new quantum dot-based method to detect specific sequences of DNA is proposed. The capture and reporter probes do not hybridize to each other, but in the presence of a template they can anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the 5' end of the template target DNA remains free to hybridize with another reporter. In this way, each capture DNA is an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This forms a superstructure, enhances base stacking, and produces a strong fluorescent signal. The introduction of T4 DNA ligase further stabilizes the superstructure and greatly increases the fluorescence intensity, and the detection limit is as low as 10 fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap. This simple technique is promising for improving medical diagnosis and treatment.     

Automated discrimination of polymerase chain reaction products with closely related sequences by software-based detection of characteristic peaks in product ion spectra

A computer-based method is described for automated detection of peaks in product ion spectra that allows discrimination of structurally related polymerase chain reaction (PCR) products. PCR products of K-ras mutants having single nucleotide substitutions and isomeric sequence changes in positions 1 and 2 of codon 12 (e.g. TGT and GTT) were used as a model system. SpecDiff, a tool for differentiating pairs of mass spectra by identifying peaks that either differ in relative intensity between spectra or only appear in one of a pair of spectra, was created to help automate detection. This program was demonstrated to have great utility in detection of mutations and could also be useful as a general tool for differentiating other molecules of closely related structure.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.)

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.     

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.