A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.     

Rapid and sensitive detection of DNA in Southern blots with chemiluminescence

A new chemiluminescent Southern blot procedure offers molecular biologists a safe, ultrasensitive and rapid alternative to conventional 32P-based systems. This new DNA detection system, SOUTHERN-LIGHT, has been developed by Tropix, Inc. The luminescent signal is produced from a direct chemiluminescent substrate, disodium 3-(4-methoxyspiro[1,2-dioxetane-3-2'-tricyclo-[3.3.1.1 .3,7]decan]-4-yl) phenyl phosphate (AMPPD), which decomposes upon dephosphorylation with alkaline phosphatase. SOUTHERN-LIGHT is an ultrasensitive, rapid detection kit for use with membrane-bound DNA. It is the first test kit to incorporate AMPPD. It requires no specialized equipment and results can be conveniently imaged on instant film or x-ray film within 5-60 min of exposure.     

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.     

The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: A comparison with radioactivity

A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2-spiroadamantane)-4-methoxy-4(3 phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.     

1,2-Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays

We have synthesized and studied two 1,2-dioxetane-based chemiluminescent enzyme substrates: 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), and, 3-(2′-spiroadamantane)-4-methoxy-4- (3″-β-D ′-galactopyrano-yloxy)phenyl-1,2-dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and βD -galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which ‘stabilizes’ the dephosphorylated AMPPD emitter. Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer. AMPPD and AMPGD offer alternatives to colorimetric and fluorescent subsrates for alkaline phosphatase and β-D -galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. β-hCG, LH, TSH and others).     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

A comparison of substrates for quantifying the signal from a nonradiolabeled DNA probe.

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.     

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.     

Improved chemiluminescent western blotting procedure.

A chemiluminescent Western blotting procedure and its application in assays for human transferrin and human immunodeficiency virus-I antibodies are described. The procedure is based on a chemiluminescent substrate, adamantyl 1,2-dioxetane aryl phosphate and alkaline phosphatase-labeled detection antibodies. Different membranes (polyvinylidene fluoride, nitrocellulose, nylon) and a proprietary membrane treatment agent (Nitro-Block) have been studied. This sensitive blotting procedure utilizing AMPPD, a polyclonal rabbit anti-transferrin:goat anti-rabbit IgG-alkaline phosphatase detection complex, and a PVDF membrane blocked with Nitro-Block permits the detection of 125 pg (1.6 fmol) of human transferrin. A novel 1,2-dioxetane substrate, CSPD, has also been evaluated.     

Resistance to ACNU induced toxicity in transgenic tobacco suspension cultures withada gene transferred fromEscherichia coli

The protein coding region of theE. coli DNA repair geneada combined with the CaMV 35S promoter has been transferred to tobacco by means ofAgrobacterium tumefaciens Ti plasmid. In transgenic plants having theada gene in a sense orientation, detectable amounts of O⁶-alkylguanine-DNA-alkyltransferase has been found whereas in non-transformed plants this activity is absent. Cell suspension cultures derived from the former plants showed lower sensitivity to the toxic (growth inhibiting) effects of the bifunctional alkylating agent 1-(2-chloroethyl)-1-nitroso-3-(aminomethyl-1,3-diazinylo)-methylurea compared with cell cultures derived from a control non-transformed plant or from transgenic plants harbouring theada gene in an opposite, non-sense orientation.     

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.     

Sorting of Glycoprotein B from Human Cytomegalovirus to Protein Storage Vesicles in Seeds of Transgenic Tobacco

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.     

Characterization of alkaline phosphatase labeled UidA(Gus) probe and its application in testing of transgenic <Emphasis Type="Italic">tritordeum</Emphasis>

Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA–enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.     

一种转基因植物高特异定性检测方法

近年来,植物基因工程技术的迅速发展使得转基因植物及其产品与人们生活越来越密切.与此同时,转基因植物检测技术不断丰富和发展,主要是在整合、转录、翻译等不同水平上检测转基因产物的存在.但各种方法都存在不同的缺点,如缺乏特异性、假阳性较高,操作繁琐、成本高等.利用TAIL-PCR(thermal asym-metric interlaced PCR)和融合克隆结合建立一种简单、快速、特异性高的转基因植物检测方法,以两个转基因植株(C3-3株系和TB-5株系)为例,通过TAIL-PCR获得其侧翼序列后设计特异引物,并用于特定转基因植株的筛选.     

Non-radioactive detection of single-copy DNA-DNA hybrids

A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.     

A colorimetric method for DNA hybridization.

A general method has been developed which allows crosslinks to be produced between proteins and single-stranded DNA. Such single-stranded DNA protein complexes have been tested for blot hybridization using two colorimetrically detectable enzymes, namely peroxidase and alkaline phosphatase, as the protein moiety of the probe. After hybridization and incubation with a substrate solution sequences complementary to the probe can be visualized directly without the need of tedious cytochemical sandwich methods. This procedure will detect target sequences, a few kilobases long, in the 1- to 5-pg range.     

Gene targeting in plants

Although the generation of transgenic plants is now routine, the integration of foreign genetic information has so far been at random sites in the genome. We now present evidence for directed integration into a predicted location in the host plant genome. Protoplasts of transgenic tobacco (Nicotiana tabaccum) plants carrying copies of a partial, non-functional drug-resistance gene in the nuclear DNA were used as recipients for DNA molecules containing the missing part of the gene. Molecular and genetic data confirm the integration of the foreign DNA through homologous recombination within overlapping parts of the protein coding region, resulting in the formation of an active gene in the host chromosome. This approach is referred to as gene targeting. The gene targeting frequency (the number of drug-resistant clones resulting from gene correction compared to the number of resistant clones from parallel experiments with a similar non-interrupted hybrid gene) was 0.5-4.2×10^-4. These experiments demonstrate the possibility of producing transgenic plants with desired modifications to a specific nuclear gene.     

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.     

Genetically engineered transgenic plants with the domain 1 sequence of tobacco mosaic virus 126 kDa protein gene are completely resistant to viral infection

In many plant RNA viruses, Domains 1, 2 and 3 are conserved in replicase proteins. In order to examine the interference of viral replication by the Domain 1 sequence, we generated transgenic plants transformed with DNA corresponding to the Domain 1 sequence of the TMV 126 kDa protein. This DNA sequence includes the TMV RNA from nucleotides 1 to 2,149, which comprises both the 5'-untranslated and methyl transferase region. The transgenic plants obtained showed complete resistance to TMV infection. The presence of the Domain 1 sequence in the plants completely prevented local necrosis in Nicotiana tabacum cv. Xanthi nc, and any systemic development of symptoms in Nicotiana tabacum Xanthi upon TMV inoculation. Most transgenic plants sustained the conferred resistance even under TMV inoculum concentrations up to as high as 1,000 microg/ml. To detect any accumulation of TMV coat protein or viral RNA in infected transgenic plants, immunochemical tests and Northern blot analyses were carried out. Neither viral RNA or coat protein was detectable in the systemic leaves of the completely resistant transgenic plants, whereas they were accumulated in large quantities in all of the control plants. Because of the conservation of Domain 1 in many plant RNA viruses, the acquisition of resistance to virus infection using the Domain 1 sequence appears to be a very effective strategy for breeding of viral resistant plants.