| With the completion of the Human Genome Project and beginning of post genome era, more and more SNPs and mutations are being uncovered and assembled into large SNP databases that promise to enable the dissection of the genetic essence of disease and drug response. The large number of SNPs provides a rich set of markers that can be used in a wide variety of genetic studies. The identification of a complex set of genes that cause a disease will require both linkage and association analyses of thousands of polymorphisms across the human genome in thousands of individuals. There is an urgent need for a fast, specific, sensitive, reliable and cost-effective method for the genome-wide polymorphism analysis. Microarray technology is one of the most promising approaches to this need. There are two major concerns in the microarray technology; first is the target labeling. Labeling is an important step in most of the microarray based target preparing protocol. It is not only time consuming, and rather expensive, but can also change the levels of targets originally present in the sample. Second is the reliability of hybridization. The melting curve of the immobilized duplex is greatly broadened and depressed which greatly reduces the fluorescence intensity difference between the perfect matched duplex and single base mismatched one. Moreover, it is difficult to normalize the hybridization conditions for a microarray because of massive number of probes and the insufficient knowledge of the hybridization reaction. The thesis focuses on these two concerns and the main contributions of this thesis are given in the following aspects:Amino-modified molecular beacon probes for immobilization were designed. They contain three parts: First, a single strand hairpin structure composed of a 16 base loop and 6 base pair stem. Second, a fluorescein in the internal location within the 5' arm is used as the fluorophore and a 3'-end [4-(4-dimethylaminopherylazo) benzoic acid] (DABCYL) is used as the quencher. Third, an amino group linked through a 20 base thymine spacer to the hairpin. The results indicate the specially designed molecular beacons retain a stable hairpin structure and a high specificity. They can be used to1. fabricate molecular beacon arrays for label-free targets detection.2. The schemes and detailed procedures for modification and activation of glass slides, polyacrylamide films and agarose films were presented. The amino group of the molecular beacon probes can react with the aldehyde group derivated from the different substrates to form a Schiff base, which can be reduced by sodium borohydride. There is a nearly linear relationship between the spotting concentration and the fluorescence intensity till 100μM for molecular beacons immobilized on the agarose films and the polyacrylamide films, which indicates higher immobilization capacities of these films. 3. Annealing properties of molecular beacon arrays immobilized on different substrates were investigated and compared. The glass slides, polyacrylamide films and agarose films immobilized molecular beacon arrays showed saturated quench efficiency in Tris-HCl buffer (pH=8.0) containing 100mM, 50mM and 10mM MgCl2, respectively; and the fluorescence intensities were 60%, 30% and 20% of their initial values, respectively. The higher annealing efficiency indicates more molecular beacon probes will be available for conformation change and contribute to the final fluorescence increments. 4. The hybridization specificity of different molecular beacon arrays in different ion strength hybridization buffers and with different target sequences were investigated and compared. The results show that the target sequences can be easily determined from the hybridization fluorescence images of the molecular beacon arrays immobilized on the polyacrylamide films and the agarose films at low ion strength (10mM MgCl2). The single nucleotide mismatch discrimination ratios were calculated from the fluorescence intensity increments. In Tris-HCl buffer (pH=8.0) c...
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