Bi-color detection of two target DNAs by non-radioactive in situ hybridization

Summary A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.This investigation was supported in part by FUNGO, Foundation of Medical Scientific Research in The Netherlands (grant nr 13-54-21)     

Nonradioactive southwestern analysis using chemiluminescent detection.

Replacing radioactively labeled probes by nonradioactive ones and detection by chemiluminescence instead of colorimetry allows a nonhazardous handling and offers the possibility of easily reprobing filters in Southwestern analysis. Using the described procedure, we were able to determine the molecular weight of DNA-binding proteins and achieve a high signal-to-noise ratio.     

Determination of carbofuran by flow-injection with chemiluminescent detection.

It was found that carbofuran enhances the chemiluminescence reaction between sodium sulphite and Ce(4+) in sulphuric acid, and this formed the basis of a flow-injection system with chemiluminescence detection for determination of carbofuran. Under optimum conditions, the enhanced chemiluminescence intensity was linear, with the concentration of carbofuran in the range 8 x 10(-8)-1.0 x 10(-5) g[sol ]mL, with a detection limit of 2.84 x 10(-8) g[sol ]mL (3 s[sol ]k). The proposed method was applied to the analysis of carbofuran in cabbage, with satisfactory results. Copyright (c) 2005 John Wiley & Sons, Ltd.     

A two-step hybridization method for chemiluminescent detection of single copy genes.

We have developed a technique for the chemiluminescent detection of single copy genes that eliminates the high backgrounds and problems with probe labeling associated with existing methods. The procedure employs a primary hybridization of single-stranded probe DNA to immobilized target DNA, a secondary hybridization with a covalently cross-linked oligonucleotide-alkaline phosphatase conjugate, followed by incubation in the chemiluminescent substrate AMPPD and detection on x-ray film. The key to the success of this method is that the primary probe contains a region complementary to the target DNA as well as to the oligonucleotide sequence of the secondary probe-alkaline phosphatase conjugate. Here we report our results using the two-step hybridization procedure to detect single copy genes from genomic Southern blots.     

Microfluidic biochip for chemiluminescent detection of allergen-specific antibodies

Protein microarrays for allergen-specific antibodies detection were integrated in microfluidic chips, with imaging chemiluminescence as the analytical technique. This paper demonstrates the feasibility of miniaturized chemiluminescent ELISA by presenting rapid, reproducible and sensitive detection of protein antibodies using microfluidics. Three different proteins, beta-lactoglobulin, peanut lectin and human IgG were immobilized via a "macromolecules to polydimethylsiloxane elastomer (PDMS) transfer" protocol and used as capturing agent for the detection of specific antibodies. A convenient and reversible procedure was used to bond the PDMS microarray substrate to complimentary SU-8/glass microfluidic reaction chambers. The hydrodynamic behaviours of the three proteins interactions within the micro-chambers were investigated to select the most efficient flowing parameters (come to terms with the assay time and performances). The use of optimized conditions led to the concomitant detection of three specific antibodies at pM level in 300 microL and using 6 min sample incubation time. Finally, sera from allergic patients were assayed using the microfluidic device modified with apple hazelnut and pollen allergen. The results obtained compared favourably with those obtained with the classical Pharmacia CAP system.     

Bisulfite modification of immobilized DNAs for methylation detection

We have developed a novel method for detecting DNA methylation status of multiple samples, in which the DNA samples were firstly immobilized on the slide and treated with bisulfite directly on the chip. In this experiment, DNAs of pUC19 plasmid were restricted by the enzymes, and ligated with a linker bearing 5'-terminal acrylamide group at the sticky ends. Using universal acrylamide gel polymerization technique, a large amount of DNAs could be immobilized on the slide. The immobilized DNAs were converted by soaking the chip in bisulfite reaction mixtures for 16 h. The probes for detection of the methylation patterns of CpG sites hybridized with the converted DNAs on the microarray, and non-specifically bound probes were cleaned by electrophoresis. We have optimized the experimental conditions of both bisulfite treatment and electrophoresis to increase sensitivity and specificity. The results were further validated by bisulfite DNA sequencing. The experiments show that the method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This novel method could be used as a convenient tool to detect the methylation status of the multiple genes for a large amount of samples in the future.     

Derivatization of biomolecules for chemiluminescent detection in capillary electrophoresis

An overview is presented on the power and drawbacks of the relatively unfamiliar chemiluminescence-based detection technique applied in analysis by capillary electrophoresis, for determining chemically derivatized biomolecules. Examples of the most common systems are given for many series of biologically active compounds as well as for some pharmaceuticals. The most common chemiluminescent systems include the application of peroxyoxalate ester chemiluminescence, acridinium esters, luminol and derivatives, detection based on the tris(2,2'-bipyridine)ruthenium(III) system, the huge potentials offered by direct oxidations-though often with still unelucidated reaction mechanisms-and the powerful area of bioluminescence techniques, revealing as well the fast developing area of microchip-based analysis employing this specific luminescence principle.     

Photochemiluminescent detection of antiradical activity. VII. Comparison with a modified method of thermo-initiated free radical generation with chemiluminescent detection.

The method of photosensitized chemiluminescence (PCL) allows the quantification of water- and lipid-soluble antioxidants and activity of superoxide dismutase (SOD) in the same measuring system. However, it needs a special device, which we have described in a previous paper in this series. Another method suitable for the assay of water- and lipid-soluble antioxidants is the thermo-initiated decay of azo-compounds combined with the measurement of O2 consumption (Niki, 1985; Wayner et al., 1985). Its long duration and the complicated measuring procedure is not acceptable for routine medical applications. We show that a modification using CL detection of free radicals with luminol, has results comparable with PCL for the determination of non-enzymic water- and lipid-soluble antioxidants, SOD activity and oxidative modification of proteins. In contrast to PCL, it is possible to use any luminometer with a heatable measuring cell and to investigate coloured samples. While the new method has an overall higher sensitivity and is scalable to microtitre plates, PCL measurements can be made at different pH. The advantages and analytical information content of certain components of the integral antioxidative capacity of blood plasma are discussed in comparison with other methods.     

Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology.

Nonradioactive detection methods for DNA, RNA, and protein analysis have been the subject of research for several years. In this paper the application of the digoxigenin nucleic acid labeling system, in combination with the new alkaline phosphatase substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl -1,2-dioxetane, to the special requirements of the analysis of transgenic plants is described. Earlier detection systems lacked the required ultrasensitive limits of detection necessary because of the large genomes found in plant cells. Routine detection of single-copy genes from transgenic plant species requires the detection of bands of picograms of specific DNA, which is easily achieved by employing the AMPPD substrate. Optimal conditions of genomic Southern analysis have been successfully adapted for Northern blotting techniques. Detection of foreign proteins in transgenic plants has proven difficult because of the very small amounts of detectable specific protein. Until now, utilization of biotinylated antibodies in combination with a streptavidin-alkaline phosphatase conjugate has been the most sensitive procedure. By introducing the AMPPD substrate, a further significant enhancement of sensitivity leading to detectable signals in the picogram range can be obtained.     

Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays

A simple enhanced chemiluminescent procedure for the quantitation of DNA hybridization to dot blots is described. The method utilizes DNA probes labeled with biotin, which are detected using a biotinylated streptavidin-horseradish peroxidase complex. The peroxidase enzyme then takes part in an enhanced chemiluminescent reaction with luminol, peroxide, and an enhancer. The method can be used to give quantitative results using a photomultiplier tube or qualitative results by recording the light emission on instant photographic film.     

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.     

Highly sensitive chemiluminescent method for the detection of cell contamination

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination. © 1998 John Wiley & Sons, Ltd.     

A sensitive CCD image system for detection of chemiluminescent reactions

A new ultra-sensitive light detection system is described for the detection of chemiluminescence reactions. The system consists of a CCD camera equipped with a two-stage image intensifier coupled to an image analysis system with real-time filtering capabilities. The sensitivity was determined with the enhanced chemiluminescence system using horseradish peroxidase as label. Additional possibilities for further increase in sensitivity and the potential use in immuoassays and other applications are discussed.     

Conformations of satellite DNAs.

X-ray fiber diffraction studies of satellite DNAs from Gecarcinus lateralis, Drosophila virilis and Mus musculus, all of which have highly repetitious base sequences but with different degrees of sequence complexity, reveal only classical polynucleotide duplex structures in contrast to some highly repetitious synthetic DNAs.