Basic Study On The Method Based On Labelling Technology With Luminescent Quantum Dots For High Through-put Screening Susceptibility Genes In Diabetes

Type 2 diabetes is a multigenic disease, resulting from a complex interaction of a number of minor gene and environmental factors. Identification and characterization of genetic variants that either cause or predispose to diabetes are a major focus of biomedical research. With the quickly development of technology of single nucleotide polymorphism, the locating of susceptibility genes of diabetes based on SNP as genetic graph has been the recent research tendency. At present, many methods for SNP genotyping have been established. As reliability, convenienc and economics as concerned, Allele-specific PCR has been used widely.The rationale of Allele-specific PCR (AS-PCR) is that the target amplification method is based on positioning the 3'base of a PCR primer to match one SNP allele. The allele-specific primers are designed and extention only happens when the primers matche with templates correctly under stringent conditions. So, the absence of the specific PCR product reveals a deviation from the wild-type DNA sequence. Multiple allele mutant site can be amplified in the same system by this method. But the observation of the results always depend on the technology of gel electrophoresis, which limits its application on high through-put creening of multiple genes SNP becaused of the poor discrimination of gel electrophoresis.Compared to high through-put and high-sensitivity detection technology, the development of semiconductor quantum dot has provided us with a new opportunity. Quantum dots with different sizes can emit multi-color fluorescence by single laser excitation. The excellent optical properties particularly apply to simultaneous analysis of multiple biological macromolecules. In recent years, in particular, the methods for surface modification and bioconjugation of quantum dot have been developed, which encouraged it's applications in the biomedical field. Quantum dot as a biological fluorescent probe has its unique advantages: water-soluble, stable, strong fluorescence, high discrimination. And it can facilitate high through-put biological detection by multi-color labelling under the same excitation light source. Researchers in related fields have conducted exploratory researches of gene expression and single nucleotide polymorphism and have made gratifying progress.Isolation and purification of DNA are usual technology involved in SNP genotyping. The traditional methods, such as recovery from gels and chromatography, have multi-steps and are time-consuming. Magnetic separation technology, developed in recent years, can quickly separate target DNA or protein from sample, without steps of centrifugation, column and filtration. It is convenient, rapid, high sensitivity and specificity. The characteristic of magnetic nanoparticles coupling with biological macromolecules is obtained by chemical surface modificantion, which provides the material basis so as to achieve efficiently separation and purification of DNA. It can also greatly improve the sensitivity and specificity SNP genotyping.In this study, we applied AS-PCR in combination with purification technology of magenitic nanoparticles and labelling technology of quantum dots to detect SNP of two susceptibility genes in diabetes in the same system.This study is divided into four parts: (1) Optimization of allele specific PCR techniques. The primers with -55C / T polymorphism site of UCP3 gene were designed. The wild-type and mutant fragments of UCP3 gene was amplified from human genomic DNA by PCR and cloned into pMD18-T vectors to construct the plasmids containing the polymorphism sites of UCP3 gene. Optimization of allele specific PCR was conducted with the constructed plasmids as templates. (2) The detection of -55C / T polymorphism of UCP3 gene and +45 T / G polymorphism of adiponectin gene and their relationship with T2DM. We applied the optimized AS-PCR to detect -55C / T polymorphism of UCP3 gene and +45 T / G polymorphism of adiponectin gene. Compared to RFLP, sensitivity, specificity and reliability of AS-PCR for SNP genotyping were assessed and RFLP is considered as a golden standard. The relationship between -55C / T polymorphism of UCP3 gene and +45 T / G polymorphism of adiponectin gene and T2DM has investigated. (3) The synthesis, modification of magnetic nanoparticles and quantum dots and their coupling with the DNA. The core-shell Fe3O4/poly (St-co-MPS) / SiO2 compound particles were prepared by fine-emulsion and St?ber method. CdTe fluorescent quantum dots were aqueous phase synthesized. They were modified with carboxyl on their surface to form nanoparticles suitable for bioconjugation with bio biological micromolecular. PCR primers were modified with biotin at 5 'end and magnetic nanoparticles were connected with streptavidin. Magnetic nanoparticles coupled with PCR products by the media of streptavidin-biotin. At the same time, the oligonucleotide probes were modified with 5 'thiol. Quantum dots were coupled with oligonucleotide probes by the reaction between thiol and carboxyl on the surface of QDs to prepare DNA probes labeled with QDs. (4) Establishment of a system for detecting susceptibility gene polymorphism in diabetes based on labeling technology with fluorescent quantum dots. With the mixed plasmids containding polymorphism site of UCP3 and adiponectin as study objects, we used diplex AS-PCR combined with molecular hybridization, magnetic nanoparticles to purify AS-PCR products, quantum dots to label oligonucleotide probes, so as to establish a system for detecting susceptibility gene polymorphism in diabetes. The conclusions obtained from this experiment are as follows:1. The wild-type and mutant plasmids of UCP3 gene were constructed successfully. Optimization of AS-PCR reactive conditions was conducted with the constructed plasmids as templates. It is confirmed that AS-PCR discrimination in SNP genotyping can be enhanced by introducing an additional mismatch at the penultimate base of 3'end of AS-PCR primers and properly increasing the annealing temperature, in combination with decreasing the level of dNTP in the reaction mixtured.2. The -55C/T polymorphism of the UCP3 gene is correlated with morbidity of type 2 diabetes indirectly. The T allele is a susceptibility factor of T2DM and related with the increase of CHOL in patients with T2DM. The +45T/G polymorphism of adiponectin gene is correlated with morbidity of type 2 diabetes. The genotype of GG is more vulnerable to develop diabetes than the genotype of TT. Compared with the RFLP, we validated that the specificity and sensitivity of detection by the optimized AS-PCR can reach the level of RFLP. It can be used as approach for screening type 2 diabetes susceptibility genes. 3. We prepared the carboxyl-functionalized magnetic nanoparticles and fluorescent quantum dots and made magnetic nanoparticles coupled with DNA of PCR products, quantum dots coupled with oligonucleotid probes respectively. By doing this, we achieved instantaneous purification of PCR products and the labeling of oligonucleotide probes with fluorescent quantum dots, which became the basis for further hybridization to detect SNP.4. We combined the AS-PCR with molecular hybridization and used fluorescent quantum dots to label oligonucleotide probes to establish a system to simultaneously detect two kinds of susceptibility genes polymorphisms of diabetes. Its specificity was confirmed in plamids mixture. It will settle a foundation for high through-put detecting susceptibility genes polymorphisms of diabetes using multi-color quantum dot labeling.Based on allele-specific PCR, the PCR products are purified by magnetic nanoparticles and hybridized with gene probes labeled with quantum dots after denature. By detecting the signals of quantum dots probes hybridized, the genotype of samples are determined. Therefore, the detection for SNP genotype of two genes at the same time has been achieved. We confirmed that using magnetic nanoparticles to purify AS-PCR products can improve the sensitivity of detection and using quantum dots to label genes probes can resolve the problem of high through-put of detection. It laid the foundation for the establishment of method of multi-color quantum dots-labeled probe screening susceptibility genes in T2DM.Combining the gene diagnosis and nanotechnology, this method make the SNP detect more convenient, economic and high through-put. It can be applied in the screening of susceptibility gene of disease, which plays an active role in early diagnosis and prevention of diseased.