Tag/hybridization-based sensitive detection of polymerase chain reaction products

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.     

Determination of Herpes simplex virus DNA with the use of solid-phase methods for the detection of PCR products

To determine DNA of herpes simplex virus (HSV), types 1 and 2, the polymerase chain reaction (PCR) method was developed with the subsequent detection of amplification products by means of electrophoresis or the molecular hybridization of nucleic acids (MHNA). Two variants of MHNA have been compared: hybridization in the solution of a biotinylated probe with digoxigenin-labeled PCR with the subsequent sorption of hybridization complexes onto streptavidin-covered plates and solid-phase hybridization of digoxigenin-labeled PCR with a biotinylated probe. Effective hybridization was observed after the denaturation of targets at 95 degrees C in the solution of 50 mM NaOH, but not in neutral solutions. To increase the level of sensitivity of hybridization in solution, the exact selection of the amount of the probe was shown to be necessary, for both its excess and deficiency essentially decreased the method sensitivity. A decrease in the ionic power of hybridization solutions from 6 h SSC to 1 h SSC led to greater specificity of hybridization without a decrease in the method sensitivity. A rise in the temperature of hybridization and subsequent washing to 45 degrees C decreased the sensitivity of the method. The limit of the sensitivity PCR with electrophoretic detection was 30 HSV genome equivalents, and 10 genome equivalents in the presence MHNA in the solution and on the solid phase.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres

Hybridization kinetics of DNA sequences with known secondary structures and random sequences designed with similar melting temperatures were studied in solution and when one strand was bound to 5 μm silica microspheres. The rates of hybridization followed second-order kinetics and were measured spectrophotometrically in solution and fluorometrically in the solid phase. In solution, the rate constants for the model sequences varied by almost two orders of magnitude, with a decrease in the rate constant with increasing amounts of secondary structure in the target sequence. The random sequences also showed over an order of magnitude difference in the rate constant. In contrast, the hybridization experiments in the solid phase with the same model sequences showed almost no change in the rate constant. Solid phase rate constants were approximately three orders of magnitude lower compared with the solution phase constants for sequences with little or no single-stranded structure. Sequences with a known secondary structure yielded solution phase rate constants as low as 3 × 10³ M⁻¹ s⁻¹ with solid phase rate constants for the same sequences measured at 2.5 × 10² M⁻¹ s⁻¹. The results from these experiments indicate that (i) solid phase hybridization occurs three orders of magnitude slower than solution phase, (ii) trends observed in structure-dependent kinetics of solution phase hybridization may not be applicable to solid phase hybridization and (iii) model probes with known secondary structure decrease reaction rates; however, even random sequences with no known internal single-stranded structure can yield a broad range of reaction rates.     

Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl₂ and primer annealing temperature of 55°C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelex™ 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was ≤10¹–10² cells following a “double” multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneComb™ (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneComb™ assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish. Received: 17 November 1997 / Accepted: 17 February 1998     

Preparing of single-stranded DNA in single-stage PCR with low-melt excess primer for hybridization on biochips

A method for fluorescently labeled single-stranded DNA (ssDNA) production during single-stage polymerase chain reaction (PCR) for subsequent hybridization on a biochip was described. This approach, whose efficiency was confirmed in the case of DARC gene, is considered as an alternative to two-stage nested PCR, consisting of two separate reactions: symmetric and asymmetric. Implementation of PCR in a single stage was achieved due to the use of a truncated excess primer in the second stage that does not anneal on the matrix during the cycles of symmetric stage of PCR and that enters the reaction after decrease of the annealing temperature in asymmetric stage. As a result, high efficiency of genotyping by means of hybridization on biochips is maintained. The suggested approach will allow us to reduce the time, working hours, and risk of contamination when researching biochips.     

In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy

Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell-localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl₂, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT-PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.     

Microdevice‐based solid‐phase polymerase chain reaction for rapid detection of pathogenic microorganisms

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab‐on‐a‐chip microdevice utilizing solid‐phase polymerase chain reaction (SP‐PCR) for point‐of‐need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP‐PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine‐modified forward primers. During SP‐PCR, the immobilized forward primers and freely diffusing fluorescence‐labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP‐PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP‐PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.     

Swine in Biomedical Research: Creating the Building Blocks of Animal Models

Abstract

Sex of preimplantation porcine embryos was determined by DNA amplification using porcine male(Y chromosome)‐specific DNA primers in the polymerase chain reaction (PCR). In order to determine the sensitivity of this sexing method, single porcine embryos ranging from unfertilized ova to the blastocyst stage were amplified in the PCR using the Y‐specific primers, and analyzed by ethidium bromide‐staining of polyacrylamide gels. The 192 bp product which denotes the presence of the Y chromosome was seen in the embryos. The unfertilized ova which is of female origin gave no product. These results are representative of PCR analysis of a total of 34 swine embryos.

Results obtained using the PCR for sexing were validated by karyotyping and confirmed by in situ hybridization with the porcine Y‐chromosome‐specific probe. In order to confirm the sex of the embryos determined by PCR, 10 day‐old porcine preimplantation embryos were biopsied to produce a small number of cells for sex determination via PCR, while the remainder of the embryo was prepared for in situ hybridization using the biotinylated probe. In situ hybridization performed on embryos shown to be male by PCR, showed pinpoint fluorescence within the nuclei, similar to that obtained when male porcine lymphocytes were hybridized. No evidence of fluorescence was seen when in situ hybridization was performed in parallel on embryos determined to be female by the PCR.

The PCR was found to be a relatively fast, accurate and reproducible means of sex determination of swine preimplantation embryos. This capability could have significant impact on animal breeding and production programs by using PCR as a screening tool for traits of economic importance.     

A PCR-ELISA Method for Direct Detection of the Oyster Pathogen Haplosporidium nelsoni

A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection. Received April 14, 1998; accepted September 30, 1998.     

Large mitochondrial repeats multiplied during the polymerase chain reaction

The number of repeats in repetitive DNA like micro‐ and minisatellites is often determined by polymerase chain reaction (PCR). When we counted repeats in an array of mitochondrial repeats in the cattle tick (Boophilus microplus) we found that the number of repeats increased during PCR. Multiplication of the repeats was independent of the primers used to amplify the region, the PCR annealing temperature and the length of the PCR product. The use of PCR to determine the number of repeats in arrays needs to be reassessed. For long repeats, a subset of samples should always be analysed by Southern blot hybridization to confirm the PCR results.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Detection and Quantification of Dehalococcoides-Related Bacteria in a Chlorinated Ethene-Contaminated Aquifer Undergoing Natural Attenuation

Detection and quantification of bacteria related to Dehalococcoides is essential for the development of effective remediation strategies for tetrachloroethene (PCE)-contaminated sites. In this study, the authors applied three methods for quantifying Dehalococcoides-like bacteria in a PCE-contaminated aquifer undergoing natural attenuation in Grenchen, Switzerland: a catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) protocol, a competitive nested polymerase chain reaction (PCR) approach, and a direct PCR end point quantification with external standards. For the investigated aquifer, multiple lines of evidence indicated that reductive dechlorination (and likely dehalorespiration) was an active process. Both PCR-based quantification methods indicated that low numbers of mostly sediment-bound Dehalococcoides were present in the contaminated zone of the Grenchen aquifer. Estimates based on the quantitative PCR methods ranged from 2.1 × 10⁷ to 1.5 × 10⁸ sediment-bound Dehalococcoides 16S rRNA gene copies per liter of aquifer volume. In contrast, the liquid phase only contained between 8 and 80 copies per liter aquifer volume. CARD-FISH was not sensitive enough for the quantification of Dehalococcoides cell numbers in this aquifer. Cloning and sequencing of the PCR products revealed the presence of sequences closely related to Dehalococcoides isolates such as D. ethenogenes and Dehalococcoides sp. BAV1. An apparently abundant group (termed “Grenchen Cluster”) of sequences more distantly related to Dehalococcoides was also identified, so far without cultured representatives.     

Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes

A simple and rapid method for characterizing the polymorphism at the HLA-DPB1 locus has been developed. The procedure involves the selective amplification of the polymorphic second exon of the DPB1 locus by the polymerase chain reaction (PCR), followed by hybridization of the amplified DNA with 15 nonisotopic sequence-specific oligonucleotide probes. There are no sequences within the second exon of the DPB1 locus that uniquely define an allele; rather, each allele appears to arise from the shuffling of a limited number of polymorphic nucleotide sequences in six regions of variability. Consequently, individual alleles are identified by the pattern of hybridization of the 15 probes. Two formats for typing are described. In Format I (the dot-blot), the amplified DNA is ultraviolet (UV) cross-linked to a nylon membrane and hybridized with the oligonucleotide probes which are covalently labeled with horseradish peroxidase (HRP). In Format II (the reverse dot-blot), the oligonucleotides, which have poly-T tails, are bound to the membrane and the immobilized array of probes is hybridized to the PCR product which has incorporated biotinylated primers during the amplification process. In both formats, hybridization is detected by a simple colorimetric reaction. The application of this technology to the fields of tissue typing and individual identity is discussed. Offprint requests to: A. B. Begovich.     

In situ localization with digoxigenin-labelled probes of tau-related mRNAs in the rat pancreas

Summary Two cDNA probes complementary to fetal rat brain tau cDNA were produced by the polymerase chain reaction (PCR) and labelled by digoxigenin-11-dUTP incorporation during the PCR elongation step. These probes were tested for thein situ localization of tau mRNAs in sections of rat cerebellum. The hybridization signal was consistent with the known localization of brain tau mRNAs, showing the validity of cDNA probes labelled by digoxigenin during the PCR. Using these probes, anin situ hybridization protocol was established and optimized for the localization of tau-related mRNAs in sections of pancreas. The aim was to determine whether these mRNAs were expressed in the exocrine or the endocrine part of the pancreas. A positive signal was found only in the exocrine part of the pancreas, and was distributed exclusively in the cytoplasm of acinar cells. The results described here are the first evidence for a specific expression of tau-related proteins in the exocrine pancreas.     

Molecular genetics of growth and development in Populus. III. A genetic linkage map of a hybrid poplar composed of RFLP,STS, and RAPD markers

We have evaluated three DNA-based marker types for linkage map construction in Populus: RFLPs detected by Southern blot hybridization, STSs detected by a combination of PCR and RFLP analysis, and RAPDs. The mapping pedigree consists of three generations, with the F₁ produced by interspecific hybridization between a P. trichocarpa female and a P. deltoides male. The F₂ generation was made by inbreeding to the maximum degree permitted by the dioecious mating system of Populus. The applicability of STSs and RAPDs outside the mapping pedigree has been investigated, showing that these PCR-based marker systems are well-suited to breeding designs involving interspecific hybridization. A Populus genome map (343 markers) has been constructed from a combination of all three types. The length of the Populus genome is estimated to be 2400–2800 cM.Abbreviations RFLP restriction fragment length polymorphism - STS sequence-tagged site - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA     

Comparison of different approaches for the incorporation of non-radioactive labels into polymerase chain reaction products

Different methods for labelling polymerase chain reaction (PCR) products with non-radioactive labels for their detection by hybridization with immobilized DNA probes were compared. The use of digoxigenin (DIG) as a label provided greater sensitivity than biotin in a PCR system targeting the invA gene from Salmonella typhimurium. Incorporation of digoxigenin into amplicons in the form of 5-DIG-labelled oligonucleotide primers resulted in better assay signals and was more economical than DIG-labelled dUTP.     

从cDNA文库中筛选分析阳性克隆的简便方法

cDNA文库中阳性克隆的传统筛选分析法既费时又费力.利用微波炉加热的方法,简化了原位杂交中噬菌斑裂解、DNA变性与固定的程序;进一步运用PCR扩增技术,特异扩增克隆载体中插入的cDNA片段,加快了阳性克隆分析的进程.     

Detection of Bacteroides fragilis in clinical specimens by polymerase chain reaction amplification of the neuraminidase gene

The polymerase chain reaction (PCR) was used in an attempt to detect Bacteroides fragilis by amplifying a segment of the gene encoding B. fragilis neuraminidase. Forty-five reference strains representing 45 species and 113 clinical isolates were tested. Only B. fragilis was PCR positive, except for Bacteroides merdae ATCC 43184, which gave a band by ethidium bromide staining that showed no signal by Southern hybridization. Using a protocol that employed DNA extraction by Sepa Gene kit and a highly sensitive digoxigenin-chemiluminescence detection system, detection of B. fragilis by PCR was in complete agreement with culture results for 44 clinical specimens from which a wide range of aerobic and anaerobic organisms and fungi were recovered.     

Physical mapping of inhibin β-A in domestic cattle

The gene for the -A subunit of inhibin (INHBA) was assigned to bovine syntenic group U13 by bovine x rodent hybrid somatic cells and the polymerase chain reaction (PCR). A 482-bp PCR fragment was used to clone a 37-kb cosmid. This cosmid was assigned to bovine Chromosome (Chr) 4 (BTA 4) by fluorescence in situ hybridization (FISH). This is the first assignment of a U13 marker to a bovine chromosome. A restriction fragment length polymorphism (RFLP) was detected with PstI within the INHBA cosmid.