The Methodology Of Genetic Mutation Detection By Non Gel Sieving Capillary Electrophoresis

It is important to diagnosis cancers in early stage for morbid cure. Genetic mutations affect the expression of genes and the function of gene products, which are thought to be the main causes of most human cancers. Genetic mutation analysis plays an important role in early clinical diagnosis of cancer. At the present time, most genetic mutation detection method is based on PCR technology. Restriction Fragment Length Polymorphism analysis is characterized by simplicity, high veracity. PCR-RFLP together with electrophoresis is often used for analyzing of genetic mutation. Slab-gel electrophoresis is characterized by low sensitivity, amplus sample and reagents and low automation. It can not be applied in clinical diagnosis cosmically.Non gel sieving capillary electrophoresis is the technique that based on the combination of electrophoresis and chromatography. It is characterized by high separation efficiency, high resolution, short analysis time, small sample. It has become an important technique in separating of DNA fragments. DNA separation in non gel sieving solutions is a complex process. Many factors such as characteristic of separation matrix, additives, pH of buffer, electrophoresis temperature, electric field strength and electroosmosis influence the migration of DNAs in separation matrix. Characteristic of separation matrix have a great effect on DNA separation by non gel sieving capillary electrophoresis.In order to achieve better application of non gel sieving capillary electrophoresis in early diagnosis of cancer, the methodology of genetic mutation detection by non gel sieving capillary electrophoresis were systematically investigated in our experiment. This paper has two parts.Synthesization and DNA separation of two different length linear polyacrylamides (LPA I and LPAII) were investigated in first part. Two LPAs were synthesized by using isopropanol as a chain transfer agent and used as separation matrix in the experiment. The differences of physical characteristic and separation power of the two LPAs were investigated. Molecular weight, entanglement threshold and gyration radius were different between two LPAs. But they had the same sieve pore under the same concentration. Both LPAs could be successfully used for separating smaller than 70bp DNA fragments of PBR322/BsuRI DNA Marker under the optimum separation conditions. At the same time, high resolution was obtained. Higher resolution of smaller than 70bp DNA fragments was obtained using lower molecular mass LPA I as separation matrix than LPA II. Shorter migration time of DNA fragments was obtained using higher molecular mass LPAII as separation matrix than LPA I . So the two LPAs can be used for mutation analyzing of small DNA fragments in clinical diagnosis and base research of cancers. A fast and sensitive analysis method for smaller than 70bp DNA fragments was established in clinical and base research. The two LPAs were tentatively used as separation matrixes to separate 1Kb DNA Marker P. 1Kb DNA Marker P was not separated completely in any separation conditions of the two LPAs.The mutations of codons 248 and 249 in p53 gene from tissue specimens of 59 gastric cancers were simultaneity detected by NGS-CE-RFLP in second part. Genomes of gastric cancer patients were extracted. The extracted genomes DNA had better concentration and purification. The DNA fragment including 248 and 249 codons was amplified by PCR. The PCR product was purified by UNIQ-10 Column DNA Purification Kit to remove salts and primer-dimer. PCR product was cleaved by Msp I and HaeIII at the same time. The optimum separation condition in separating smaller than 70bp DNA fragments was applied in analyzing the mutation of codons 248 and 249 in p53 gene. The optimum separation condition was 8%LPA I with 8%mannitol, running pH at 6.5, running temperature at 15℃and electric field strength at 275V/cm. Mutation detection was completed in about 30min. Baseline separation and higher resolution (1.642) were obtained in separating 35bp and 40bp DNA fragments. A fast, high resolving power method was established in clinical diagnosis of gastric cancer.In our experiment two LPAs were synthesized and used as separation matrix in capillary electrophoresis. The differences of the two LPAs in separating DNA were investigated. LPA I was used for mutation detection of p53 gene from tissue specimens of gastric cancer. A fast and sensitive analysis method for smaller than 70bp DNA fragments was established in clinical and base research. At the same time, a fast, high resolving power method was established in clinical diagnosis of gastric cancer.