Study On The Method Of Gene And Relative Protein Analysis Of Lung Cancer By Capillary Electrophoresis

Cancer is made of multi-factor and multi-stage evolution. With the development of a variety of detection technology and penetration of interdisciplinary, some biological indicators of tumor are developed from initial molecular biology detection to chemical detection. In this paper DNA and protein of lung cancer are analyzed by analytical chemical detection method of capillary electrophoresis (CE), and a new sieving medium of polyethylene oxide (PEO) containing gold nanoparticles (GNPs) is studied.In the first part, from the DNA point of view the classic mutation-point analytical method of single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) combined with polyacrylamide gel electrophoresis (PAGE) and CE is used to detect the mutation of p53 gene exon 7. Comparing the advantages of four methods from four respects:inject volume, time, detection limit and detection rate. The results reveal that the order is:CE-SSCP>PAGE-SSCP>CE-RFLP>PAGE-RFLP. CE-SSCP can offer a convenient and reliable method to a large scale of early diagnosis of lung cancer.In the second part, the protein detection method of non-gel sieving capillary electrophoresis with laser induced fluorescence (NGS-CE-LIF) is established firstly. Then the method is improved to detect the protein complex (denatured/native) differences of lung cancer tissue and normal tissue. This method can separate protein under the range of 6.5～200 kDa. The method has the advantages of easy concentration adjustment, good separation efficiency, and short separation time. The average theoretical plate number (N) is 9.12×104 /m, and the detection limit is 2.8×10-4 mg/ml. Comparing with adjacent normal tissues, lung cancer tissues have more visible protein types and expression differences, and mainly concentrate in 20～116 kDa. Comparing with the SDS polyacrylamide gel electrophoresis (SDS-PAGE) and the Blue Native polyacrylamide gel electrophoresis (BN-PAGE), the NGS-CE-LIF is rarely the same, and CE-LIF can detect which the PAGE can't.In the third part, GNPs of 13 nm is synthesized and the size of GNPs is measured by transmission electron microscopy (TEM). The PEO-GNPs-TBE is used as the CE sieving medium. The impact of parameters:sieving medium concentration, separation voltage, temperature and sieving medium pH on DNA fragments separation are studied. Separation theories such as Ogston model and partial crawling model are used to explain the relative experimental phenomenon and preliminary study the separation mechanism. The results reveal that adding GNPs to the sieving medium can improve the separation efficiency of capillary electrophoresis. The separation effect of the new sieving medium is good and the separation time is short. The sieving medium is suitable to separate wide range of DNA fragments (100～10000 bp). The electrophoretic mobility of DNA molecule is inversely proportional to size, and shows a certain linear relationship. The linear relationship y=-kx+b can better explain the separation mechanism of this sieving medium.The application of CE in DNA and protein analysis has the advantages of highly efficient, rapidly and highly sensitivity. The application of the new nano-material provides a possible for the improvement of detection method and detection efficiency. The previously basic work can provide scientific basis for various technologies such as chips, mass spectrometry combining used and tumor detection.