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.     

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.     

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).     

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.     

A protocol for non-radioactive DNA labelling and detection in the RFLP analysis of rice and tomato using single-copy probes

An improved protocol for non-radioactive labelling and detection of rice and tomato DNA is described. The procedure includes the use of polymerase chain reaction for the incorporation of digoxigenin-dUTP in the DNA molecule and the use of a chemiluminescent compound (AMPPD) for the signal detection.     

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.     

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.     

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.     

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.     

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.     

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.     

基于地高辛标记对小麦进行Southern杂交分析主要影响因素的优化和验证

以转基因小麦和野生型小麦DNA为材料,对利用地高辛标记对小麦基因组DNA进行Southern杂交分析的影响因素进行了优化研究,包括探针制备与纯化、样品DNA量、酶切体系、真空转印条件、杂交条件、免疫检测方法等。结果表明,对随机引物标记的模板和标记后的探针进行纯化可明显提高探针的标记效率,10μg高质量的DNA样品在80μl的体系中,酶切8～12h可获得良好的效果;真空转膜时使用碱性液比中性液获得的转膜效果更干净;试剂纯度、杂交温度及杂交炉转速等均对杂交效果产生重要影响;配合改进的CSPD涂布方法,使用化学发光检测系统比单纯使用X光片显像更易操作,背景更干净;本研究所优化的地高辛标记的小麦Southern杂交分析显示出较高的灵敏度和信噪比,结果稳定,可克服同位素标记对实验条件、设备及实验人员身体状况等限制,在普通实验室推广应用。     

Fingerprinting Analysis of Saposhnikovia divaricata using 1H Nuclear Magnetic Resonance Spectroscopy and High Performance Liquid Chromatography

The 1H nuclear magnetic resonance (1H NMR) fingerprints of fractionated non-polar and polar extracts (control substance for plant drug [CSPD] A and B) from the roots of 12 specimens of Saposhnikovia divaricata (Turcz.) Schischk were achieved with Fourier Transform (FT)-NMR spectrometer and assigned by comparison to each other and to the 1H NMR spectra of the isolated individual compounds. These fingerprints were found to be uniform in terms of the specificity for the implication of all 12 specimens being systematically of the same origin. The uniformity was further affirmed by high performance liquid chromatography (HPLC), which also revealed exactly identical specificity for the identified S. divaricata species with the 1H NMR appearances of corresponding CSPD on the part of the composition of characteristic constituents when comparing to corresponding individual compounds. This investigation unambiguously shows that the specific signals from the chemotaxonomically significant compounds of chromones and coumarins in S. divaricata are exhibited distinctively in the composite features of both 1H NMR fingerprints and HPLC profiles. The 1H NMR and HPLC profiles established can successfully be used as reference for the authentication of the origin of S. divaricata species as well as for chemotaxonomic studies.     

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.     

Imaging of chemiluminescent signals with cooled CCD camera systems

We investigated imaging of chemiluminescent signals from 1,2-dioxetanes with cooled CCD cameras. Non-radioactive detection methods for biomolecules utilizing these chemiluminescent substrates for alkaline phosphatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, immunoblotting, ELISA methods and DNA sequencing. Dephosphorylation of the dioxetane CSPD by alkaline phosphatase generates an unstable anion that decomposes resulting in light production. The wavelength of the emitted light is approximately 460nm. We have utilized Photometrics Star and MXC 200L cooled CCD cameras for direct imaging of chemiluminescent signals. Benefits of utilizing a CCD detector include rapid data digitization and more accurate quantitation of chemiluminescent signals compred to film-based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our results indicate that the digitization of chemiluminescent signal data with cooled CCD cameras is an excellent alternative to ³²P detection methods utilizing storage phosphor screen imaging systems.     

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.     

Natural variability and diurnal fluctuations within the bacteriophage population of the rumen.

To investigate the impact of nutritional and environmental factors on bacteriophage activity in the rumen, it is first valuable to determine the extent of natural variations and fluctuations in phage populations from different animal species, and from animals located together and separately, and variation in animals over time. Differences in phage populations between sheep on different diets, between sheep and goats, and within the rumen over time were investigated by using pulsed-field gel electrophoresis and comparing total phage DNA in ruminal fluid. It was found that no two individuals had similar DNA banding patterns, even when similarly fed and penned together, indicating there is considerable individual diversity in phage populations between animals. Despite these individual differences, the quantities, but not the banding patterns, of phage DNA were similar for animals within groups but varied between groups, suggesting that nutritional factors may influence overall phage activity in the rumen. In sheep fed once daily, a distinct diurnal variation in the phage population was observed. Two hours postfeeding, total phage DNA dropped to its lowest level. The phage population then increased, reaching a maximal level 8 to 10 h postfeeding before declining over the next 4 h to reach a stable concentration for the rest of the cycle. The general trend in phage DNA concentration appeared similar to previously recorded diurnal fluctuations in ruminal bacterial populations in cattle fed once daily.     

Orientation of DNA on a surface from simulation

DNA orientation near surfaces determines many properties related to hybridization efficiency. We performed a 40-ns molecular dynamics simulation to study the structure and orientation of a 12-base-pair DNA duplex tethered to a neutral, epoxide-coated silica surface. Starting with a canonical B-form tethered in an up-right position, normal to the surface, the DNA tilted to over 55 degrees and back. The time scale was a few nanoseconds for tilting events. The linker between the DNA and the surface went from standing upright to tilted, and finally collapsed on the surface. Although the DNA conformation fluctuated, it remained closed to B-form for the entire 40 ns. Calculations of helical parameters of the DNA show that the tethered end of the DNA changed its conformation noticeably when attracted to the surface.     

DNA degradation in fish: Practical solutions and guidelines to improve DNA preservation for genomic research

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.     

A 300 MHz and 600 MHz proton NMR study of a 12 base pair restriction fragment: investigation of structure by relaxation measurements.

The 1H NMR spectrum of a 12 base pair DNA restriction fragment has been measured at 300 and 600 MHz and resonances from over 70 protons are individually resolved. Relaxation rate measurements have been carried out at 300 MHz and compared with the theoretical predictions obtained using an isotropic rigid rotor model with coordinates derived from a Dreiding model of DNA. The model gives results that are in excellent agreement with experiment for most protons when a 7 nsec rotational correlation time is used, although agreement is improved for certain base protons by using a shorter correlation time for the sugar group, or by increasing the sugar-base interproton distances. A comparison of non-selective and selective spin-lattice relaxation rates for carbon bound protons indicates that there is extensive spin diffusion even in this short DNA fragment. Examination of the spin-spin relaxation rates for the same type of proton on different base pairs reveals little sequence effect on conformation.