The Method And Exploratory Development Of Gene Mutation Analysis By High Performance Capillary Electrophoresis

Gene mutation always reduce tumorigenesis and they have correlation with each other . Other domain experts think that explore new and sensitive method of gene mutation detection is focus and cross science.There have many classical methods on detection gene mutation ,such as agarose and polyacrylamide gel electrophoresis. Restriction fragment length polymorphism(RFLP) and single strand conformation polymorphism(SSCP) are commonly used in gel electrophoresis. RFLP and SSCP are not sensitive and have high positive rate in gel electrophoresis. But these two techniques became sensitive and high resolution as they connect with high performance capillary electrophoresis. Explore method of gene mutation detection by capillary electrophoresis is profit to exact diagnoses and prewarning of tumor. The purpose of this dissertation is to develop a series separation systems of tumor gene mutation detection of RFLP and SSCP by CE , and our discussion on related mechanism will provide rationale for clinical diagnosis.This dissertation have three parts, first part is research development, second part is about developing CE separation system of RFLP and discussing mechanism and third part is about developing CE separation system of SSCP and discussing mechanism.First part. This part reviewed development tumor relative and gene mutation. The development of tumor relative include oncogenes, anti-oncogenes and tumor biomarkers. The development of analysis of gene mutation include SSCP and RFLP.Second part. This part is about established of separation system of CE-RFLP. This part purpose is to establish pBR322/BsuRI separation system and combine self-made short line polyacrylamide (SLPA )with self-modify capillary column. The method is about research effects of SLPA concentration, temperature, voltage , mannitol and pH on double-strand DNA analysis. The results show DNA resolution rise in earlier and then degrade when SLPA concentration rise. 25℃ and 13kV is optimum condition. The properties of the separated buffer were changed by the reaction between mannitol and boric acid, which improved the resolution of double-strand DNA. The optimum concentration of mannitol is 8%. Low pH can increase volume of sample injection, diminish width of peak, but the resolution is lower thanSLPA contain mannitol. The separation system can detect from 20 base pairs to 600 base pairs. The experimental data show that common logarithm of DNA mobility has linear correlation with concentration of SLPA, and PO.01, so the mechanism is Ogston pattern. And we have developed a cubic equation and it's independent is length of DNA , variable is migration time. The equation can help choosing restriction enzyme in RFLP.The third part. This part is about establishment of CE-SSCP. The purpose is to establish single strand DNA separation system. Method is that We use the ampicillin resistance gene of pBR322 and pUC19 as point mutation model and we investigated effects of the SLPA concentration , temperature and voltage .Results show temperature is most important on DNA analysis ,and optimum temperature and SLPA concentration is 19°C and 6%,respectively. Resaults show that the model is reasonable and single strand separation system is reliable, the model can be used to investigate other single-strand separation system for it's rationality and known sequence . MFOLD is a software which can help designing antisensenucleic acids drugs. We must know secondary structures of nucleic acid when we want to design antisensenucleic acids drugs .We used MFOLD to predict single-strand secondary structures, and results of this prediction have three structures are not same as four peaks in actual electropherogram. Maybe it has two reasons. The first is that parameters of MFOLD is different with true electrophoresis.The second is we suppose the true reason of four peaks in electropherogram is not single-strand secondary structures but is more complicated structures.