Biotinylated Polymerase Chain Reaction Products Research Articles

Cloth-based closed bipolar electrochemiluminescence DNA sensors (CCBEDSs): A new class of electrochemiluminescence gene sensors

In this work, for the first time, the cloth-based closed bipolar electrochemiluminescence (CBP-ECL) DNA sensors (CCBEDSs) have been presented as a new class of electrochemiluminescence gene sensors, where the integration of magnetic immobilization/enrichment and CBP-ECL can be served for facile detection of double-stranded polymerase chain reaction (PCR) products of Salmonella enteritis (S. enteritis) invA gene. A self-made support is used to combine avidin-conjugated magnetic beads (AMBs) with the CBP-ECL cloth-based device, where the magnet adsorbs the AMBs onto the anode of closed bipolar electrode (C-BPE) to well immobilize and enrich the biotinylated PCR products. And, the Ru(bpy)32+ (TBR) molecules are cast onto the anode surface. When the tripropylamine (TPA) is applied to the sensing interface of DNA sensors, the TBR shows the excellent electro-photo properties. Under optimized conditions, the ECL intensity was found to be logarithmically related to the concentration of PCR products in range from 0.001 to 1000 pM. The limit of detection is 0.05 fM (about 29.93 copies/μL) and the relative deviations are 2.2% and 3.9% for inter- and intra-assays at 50 pM PCR products. The results show that the CCBEDSs have the relatively good sensitivity, wide linear range, high specificity and good stability, and have no any complex purification steps for amplification products. In addition, the CCBEDSs can determine S. enteritis in milk, egg white, grated tomato and beef samples. Therefore, the method may have broad application prospects in medical diagnosis and food detection.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

Development of an immunoFET biosensor for the detection of biotinylated PCR product.

ImmunoFET (IMFET) biosensor is a simple platform for the detection of biotinylated products of polymerase chain reaction (PCR). Construction of the IMFET biosensor started with adsorption of 1.5 mg/mL of protein A (PA) onto the insulated gate surface of ISFET for 90 min. Next, the immobilized 1/500 dilution of anti-biotin antibody was adsorbed onto the PA layer for 60 min. The IMFET biosensor was subsequently ready for detection of the biotinylated amplicon. The IMFET biosensor showed highly specific binding to the biotinylated PCR product of the phaE gene of Haloquadratum walsbyi DSM 16854. The phaE gene is a biomarker of polyhydroxyalkanoate (PHA) producers that contain PHA synthase class III. The lowest amount of DNA template of H. walsbyi DSM 16854 that the IMFET biosensor could detect was 125 fg. The IMFET biosensor has a lower amount of detection compared with a DNA lateral flow biosensor from our previous study. The degree of linearity of the biosensor signal was influenced by the concentration of the biotinylated amplicon. The IMFET biosensor also has a short response time (approximately 30 times) to detect the phaE amplicon compared to an agarose gel electrophoresis. The IMFET biosensor is a promising tool for the detection of the biotinylated PCR product, and it can be integrated into a micro total analysis system (μTAS).

Affinity capture and solid-phase sequencing of biotinylated PCR products.

The time and labor required for generating nucleotide sequence information has been significantly reduced since the development of the polymerase chain reaction (PCR). PCR allows the production of sufficient amounts of DNA template in vitro, and consequently, the time-consuming cloning procedure to obtain large enough quantities of DNA for sequencingisavoided. The PCR has transformed nucleotide sequencing into a method that can be considered even in routine diagnostics for sequencing large numbers of individual samples.

Identification of Blood Meals Imbibed by Phlebotomine Sand Flies Using CytochromebPCR and Reverse Line Blotting

Blood meal identification is important for determining the host preferences and the vectorial capacity of hematophagous arthropods. In the past, mostly serological techniques using host-specific antibodies were used, but in recent years more sensitive and accurate polymerase chain reaction (PCR)-based molecular approaches for identifying blood meals have been developed. Here, a vertebrate-specific PCR is combined with reverse line blot analysis for identifying blood meals ingested by female phlebotomine sand fly vectors of leishmaniasis. Species-specific oligonucleotides were covalently linked to nylon membranes, and biotinylated PCR products of the mitochondrial cytochrome b gene were used as probes in a hybridization reaction revealed using colorimetric or enhanced chemiluminescent detection systems. This combination identified blood meals up to 96 hours after ingestion containing minimal amounts of DNA (>0.1 pg). The specific probes discriminated between putative host species in several study areas. The source of blood was identified in 68 of 89 wild-caught sand flies tested (76%). Mixed blood meals were identified in 15 (17%) of those. The advantages and limitations of this method are discussed.

Electrical DNA-chip-based identification of different species of the genus Kitasatospora

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 mug/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.

Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

A SIMPLE, HIGH-THROUGHPUT METHOD TO DETECT PLASMODIUM FALCIPARUM SINGLE NUCLEOTIDE POLYMORPHISMS IN THE DIHYDROFOLATE REDUCTASE, DIHYDROPTEROATE SYNTHASE, AND P. FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER GENES USING POLYMERASE CHAIN REACTION– AND ENZYME-LINKED IMMUNOSORBENT ASSAY–BASED TECHNOLOGY

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.

Liquid-phase hybridization based PCR-ELISA for detection of genetically modified organisms in food

Polymerase chain reaction (PCR) screening for presence of transgenic components in food is becoming a routine method in modern food analysis. To develop a high throughput method for quantitation of the PCR products is needed for automatic industry analysis. Here we described an in situ liquid-phase hybridization (LPH) for PCR-enzyme linked immunoabsorbent assays (PCR-ELISA) that was widely used for quantitation of PCR products. In LPH-PCR-ELISA, the biotinylated PCR product was hybridized with digoxigenin-labeled probes in the PCR reaction mixture immediately after PCR cycles and the hybridizations was incorporated into the PCR program. Subsequent enzyme conversion of substrate gave distinct OD values when detecting samples with genetically modified organisms (GMOs) labels in the different concentrations. The described method enabled a fast, specific, and accurate detection of GMOs components in food products and thus can be developed to a full-automatic method for routine analysis of raw and processed food products in large sample number.

PCR-ELISA for the CaMV-35S promoter as a screening method for genetically modified Roundup Ready soybeans

Screening of food components using a polymerase chain reaction (PCR) for the presence of genetic elements, such as the widespread cauliflower mosaic virus 35S promoter introduced into genetically modified organisms (GMOs), has become a routine method in modern food analysis. With the aim of developing a high throughput method suitable for automation we established a PCR-enzyme-linked immunosorbent assay (PCR-ELISA). It is based on specific hybridization of an immobilized, biotinylated PCR product with a digoxigenin-labelled internal probe; the label then serves in colorimetric immunodetection. With this fast and convenient method laborious blotting procedures and the use of hazardous ethidium bromide in gel staining are avoided. The optimized protocol for this PCR-ELISA allows the detection of as little as 0.1 ng amplicon in only 2 h. With this new technique we analyzed whole Roundup Ready soybeans as well as soybean flour with GMO contents ranging from 0.1% to 2%.

Quantification of GABA A receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology

We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of γ-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA A receptor subunits: α1; α6; β2; β3; γ2 and δ. The method is quantitative and rapid. With a large dynamic range from 10 fg to 1 ng of subunit mRNA, the accuracy was 12 and 19% (intra- and interassay coefficients of variation, respectively), which might be improved by using a smaller range of standards. The use of a double logarithmic standard curve [log (standard to competitor signal) vs. log (standard mRNA originally present)] requires only one reaction from each sample, allowing the analysis of a large number of samples in one experiment.

Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization

Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (≥5·10 3 colony forming units [cfu]/100 ml) and Pectinatus frisingensis (≥5·10 5 cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

Quantitative polymerase chain reaction based on a dual-analyte chemiluminescence hybridization assay for target DNA and internal standard.

We have developed a dual-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR). The method allows simultaneous determination of both amplified target DNA and internal standard (IS) in the same reaction vessel. The target DNA from the sample (233 bp) was coamplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DNA, differing only by a 24-bp sequence, centrally located. Biotinylated PCR products from target DNA and IS were captured on a single microtiter well coated with streptavidin. The amplified target DNA was hybridized with a digoxigenin-labeled specific probe, and the hybrids were determined by using antidigoxigenin antibody labeled with aequorin. The amplified DNA IS was hybridized, in the same well, with a fluorescein-labeled probe, and the hybrids were determined by using an antifluorescein antibody conjugated to alkaline phosphatase. Aequorin was measured by adding a Ca(2+)-containing light-triggering solution. Alkaline phosphatase was measured by using a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear range extended from 430 to 315,000 target DNA molecules. Average CVs ranged from 7 to 17%. The proposed system is expected to facilitate the automation and routine use of quantitative PCR.

Detection of mutations in PCR products from clinical samples by surface plasmon resonance.

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed.

Quantification ofCoxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA)

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination of Coxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate. Biotinylated PCR product was hybridized to the immobilized capture probe. Bound product was detected via streptavidin horse-radish peroxidase. The devised nonisotopic technique allows specific, rapid, and convenient quantification of C. burnetii DNA. Additionally, it is compatible with standard laboratory ELISA equipment, making this assay amenable to automation and permitting processing of large sample numbers.

Variants within the 5′-flanking regions of bovine milk protein genes: I. κ-casein-encoding gene

In order to identify DNA variants within the 5'-flanking region of the bovine κ-casein (κCn)-encoding gene, this area of the gene from 13 cows belonging to seven breeds (Holstein Friesian, Brown Swiss, German Simmental, Jersey, Galloway, Scottish Highland and Ceylon Dwarf Zebu) was analysed. For each individual, about 1 kb of the 5'-flanking region including exon I was amplified by polymerase chain reaction (PCR). The biotinylated PCR product was immobilized on magnetic beads followed by direct bidirectional sequencing using an automated DNA sequencer. Fifteen DNA variants were identified, some of which are located within potential regulatory sites and possibly involved in the expression of the κ-casein encoding gene.

A mouse Chromosome 11 library generated from sorted chromosomes using linker-adapter polymerase chain reaction

We describe the generation of a mouse whole chromosome library using sequence-independent polymerase chain reaction (PCR) to amplify sequences contained in DNA extracted from flow sorted chromosomes. DNA in sorted chromosomes from a human x mouse hybrid cell line was digested with a frequent four-cutter restriction enzyme, Sau3AI, and the ends were ligated to an adapter oligonucleotide. The ligated DNA fragments were amplified using PCR primers homologous to the linker-adapter oligonucleotide. PCR-generated products were characterized by gel electrophoresis. The size of the amplified DNA ranged from 100 to more than 1,000 bp with a relatively high proportion of products at approximately 400 bp. Biotinylated PCR products used for FISH showed specific hybridization to murine metaphases and no hybridization to human lymphocyte and hamster metaphase cells. Banding analysis indicated that the probes were specific for mouse Chromosome 11. We anticipate that availability of painting probes for specific murine chromosomes will facilitate cytogenetic studies in the mouse.

Quantitative Analysis of Polymerase Chain Reaction Products Using Biotinylated dUTP Incorporation

A method for relative quantitation of specific mRNA species by polymerase chain reaction (PCR) has been developed by using the incorporation of biotinylated dUTP. Transferred biotinylated PCR products gave a sensitive colorimetric signal which could be quantitated by video analysis. In the exponential phase of amplification, the linearity and reproducibility of reverse transcription and PCR demonstrated the same efficiency of cDNA synthesis and PCR for the two target genes, ICAM-1 and β-actin, and allowed the normalization of ICAM-1 expression. These results suggested that in the exponential phase of amplification a relative quantitation of mRNA could be determined. We used this approach to analyze the different effects of TNF-α, IL-1β, and purified porcine platelet-derived growth factor stimulations on ICAM-1 expression in smooth muscle cells.

A Highly Sensitive and Fast Nonradioactive Method for Detection of Polymerase Chain Reaction Products

A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.

Synthesis in vitro and application of biotinylated DNA probes for human papilloma virus type 16 by utilizing the polymerase chain reaction

The polymerase chain reaction (PCR) has been used to incorporate biotinylated deoxynucleotide triphosphate analogues into a 120 bp sequence from the E6 region of human papilloma virus type 16 (HPV 16). No loss of amplification efficiency is observed utilizing concentrations of up to 200 microM-biotin-11-dUTP, or 180 microM-biotin-7-dATP and -biotin-16-dUTP (where the numbers refer to the number of carbon atoms in the spacer arms). Internally biotinylated PCR products can be detected following slot-blot or vacuum transfer to mitrocellulose or nylon filters without prior electrophoretic separation of the reactants, since unincorporated biotinylated analogues pass through the filter. Internally biotinylated PCR products can also be applied as hybridization probes in Southern blot analysis or in situ hybridization. This system enables detection of PCR products or target sequences at levels below that for 5'-biotinylated probes and can be applied in an 'open sandwich assay' without the need for a separate labelled probe currently required in conventional sandwich assays. However, as hybridization probes, sensitivity may be limited by the steric hindrance of strand hybridization possibly due to the spacer arms linking the nucleotides to the biotin molecule.