A General Route to Diverse Mesoporous Metal Oxide Submicrospheres with Highly Crystalline Frameworks

通讯作者地址: Wang, JF (通讯作者), Chinese Univ Hong Kong, Dept Phys, Shatin, Hong Kong Peoples R China 地址: 1. Chinese Univ Hong Kong, Dept Phys, Shatin, Hong Kong Peoples R China 2. Univ Calif Santa Barbara, Dept Chem & Biochem, Santa Barbara, CA 93106 USA 电子邮件地址: [email protected], [email protected] NSF 0233728 NASA URETI NCC-1-02037 HK RGC Research 2060332 NSFC/RGC joint Research Scheme N_CUHK448/06 290031

Hybridization Assays Using an Expressible DNA Fragment Encoding Firefly Luciferase as a Label

We report the use of a new label, an expressible enzymecoding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O 2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n ) 4). The specific and strong interaction between two complementary nucleic acid strands forms the basis for the development of hybridization assays. Hybridization methodology is emerging as the most promising area in laboratory medicine and has transformed the way clinical testing is realized. Previous tests have been based on the monitoring of gene products, i.e., phenotypic markers, such as oncoproteins, viral antigens, etc. In contrast, current laboratory tests that are based on hybridization allow the analysis of disease at the nucleic acid level. Thus, pre-or postnatal diagnosis of genetic disease can be accomplished by hybridization of the patient's DNA with allele-specific oligonucleotide probes that recognize mutations, deletions, or insertions causing the disease. Also, the various infectious agents can be measured in biological fluids by hybridization with specific probes. In forensic science, hybridization of DNA with minisatellite probes allows the unique identification of individuals (DNA fingerprinting). 1,2 Radioactive probes (usually labeled with 32 P), in combination with autoradiographic detection, dominated in the field of hybridization assays for more than 2 decades and provide the highest sensitivities. However, the short half-life of 32 P, the health hazards and problems associated with its use and disposal, and the long exposure times (many hours to days) required for detection have placed limitations on the routine use of hybridization assays in the clinical laboratory. The current trend in this area is toward novel nonradioactive alternatives. 2,3 The labels can be incorporated into the probes either enzymatically (e.g., using DNA polymerase or deoxynucleotidyl transferase and modified deoxynucleoside triphosphates) or by chemical conjugation (e.g., introduction of NH 2 groups into the probe via cytidine transamination and then conjugation to the reporter molecule). 4 Nonisotopic hybridization assays based on fluorescent, chemiluminescent, or enzyme labels have been developed. Generally, there are two strategies for the analysis of hybrids. Either the reporter molecule is directly conjugated to the probe, 5,6 or a ligand is attached to the probe and the hybrids are measured in a subsequent step by adding a specific, labeled binding protein. The ligand may be biotin or a hapten (e.g., digoxigenin). Labeled (strept)avidin or antihapten antibodies may then be employed for detection. 7,8 Enzymes (such as alkaline phosphatase and horseradish peroxidase) are the most widely used nonradioisotopic labels because they provide amplification through the high turnover of their substrates to detectable products. 2 Recently, we reported 9 that a DNA fragment (DNA template) coding for an enzyme can be used as a novel label for the development of highly sensitive immunoassays (expression immunoassays). In these assays, after completion of the immunoreaction, the DNA template (a luciferase-coding DNA) is expressed by in vitro transcription/translation and the activity of the synthesized enzyme is measured. Furthermore, it was estimated that 12-14 luciferase molecules were synthesized from each DNA template molecule. In the present work, we extend our investigation in the area of hybridization assays. EXPERIMENTAL SECTION Instrumentation. Luminescence measurements were carried out using a liquid scintillation counter (Model LS-6500, Beckman Instruments Inc., Fullerton, CA) in the single photon monitoring mode. Fluorescence measurements were performed with th

Internet Electronic Journal of Molecular Design

Based on the structure information of the mathematical characteristics of the degree distribution of saturated hydrocarbons, a method to count the branching number of a saturated hydrocarbon is proposed and then extended to general molecules. In order to understand the structure information of the characteristics, some new concepts are introduced, and the edges in a molecule are partitioned into different cases. In the understanding, the edges that are essential to connecting the cycles are not considered as the branches. Subsequently, several formulas are tried to calculate the proposed branching number. Then, the formulas are generalized to other molecules with poly–cycles, heteroatoms and/or multiple bonds. Most of molecules are counted in agreement with intuitive view, but there also exits some molecules that are difficult to judge the branching numbers. Th