Quantitation And Point Mutation Detection Of P53 MRNA Of Tumor Cells Using Molecular Probes

As one of the diseases that threaten human being health, malignant tumors have been studied extensively. A considerable amount of molecular biology data indicates that carcinogenesis process is accompanied by mRNA expression level change of tumor-associated genes. It is essential to establish a quick, accurate and sensitive method for detecting mRNA expression level. In this thesis, A series of molecular probes were developed to detect mRNA expression level of tumor suppressor gene p53 in total RNA quantitatively and mRNA point mutation of tumor cells. This thesis is composed of the following three parts:1. A p53 molecular beacon (p53 MB) was designed according to the base sequence of tumor suppressor gene p53 to detect p53 mRNA in extracted total RNA of tumor cells quantitatively and quickly. The specificity of the p53 MB was tested by hybridizing the MB with total RNA extracted from nasopharyngeal cancer cell lines (CNE2) and CNE2-p53RNAi cell lines in which p53 mRNA expression was reduced by RNA interference. A p53/cDNA standard curve was established to quantify p53 mRNA expression level in various cell lines. The results were in consistent with that obtained from traditional reverse transcription polymerase chain reaction (RT-PCR) method. The proposed method is simple, sensitive and suitable for detecting p53 mRNA expression level in various kinds of cell lines.2. Based on the above MB quantitative assay, the p53 mRNA expression level in lung adenocarcinoma cells (A549) treated with 5-fluorouracil (5-FU) for different time was studied. The reliability of MB detection results was further proved by classical molecular biology methods. The results showed that p53 mRNA expression level in tumor cells was upregulated by 5-FU. The highest expression level of p53 mRNA was observed when the cells treated with 5-FU for 6 hours, then the expression level downregulated for longer treatment time. The method can provide information about the influence of drugs on p53 mRNA expression level in the cells rapidly and accurately. It is expected to provide reasonable drug selectivity for the individualization of cancer treatment quickly and accurately.3. A new method for mRNA point mutation detection using fluorescence resonance energy transfer pairs was developed. Two kinds of nucleic acid probes were designed. One was labeled with tetramethylrhodamine (TAMRA) and the other with 4-[4'-dimethylamino-phenylazo] benzoic acid (DABCYL). The results showed that single base mutation could be identified. The p53 mRNA of tumor cells was also...