The Establishment Of RFLP-microchip System In Detecting Gene Mutation

PrefaceRecent advances in research of the molecular biology of cancer have identified a close relationship between oncogenes,and suppressor genes(such as APC,K-ras,p53, and DCC),so gene detection could observe the development and progression of cancer effectively.Nowadays measuring the sequence is golden criteria of gene detection,but its disadvantages such as time wasting and high cost make it impossible to using abroad.The common methods of detecting gene mutation are PCR-PFLP,PCR-SSCP,methylation measure and instability of satellites.PCR-PFLP is familiar method for detection.It has advantages such as highly repetition and simpleness and has been widely used.On the base of PCR,Imost of the method can effectively detect mutation associated with electrophoresis.However the low sensitivity of normal electrophoresis causes the increase of fake negative and localization of detecting mutation of gene.Miniaturized Total Analysis Systems is based on Microelectromechanical systems. Remarkable progress has been made in the development of microfabricated systems for biological and chemical analysis in recent years.An integrated microfluidic device incorporates many of the necessary components and functionality of the typical room-sized laboratory on a small chip.These devices are also called "lab-on-chip" systems.Microfluidic chip is a new technology that promises a way to achieve fast and highly efficient separations in microscale analytical devices.It offers several advantages over traditional gel electrophoresis,the most important of which are high-throughput capabilities,reduced sample and solvent consumption,increased portability,and reduction in analysis time.In the area of clinical and molecular diagnostics,the latter two are highly significant because they create the opportunity to develop a portable point-of-care device that could allow rapid diagnosis of various disease states.Over past decade,the dectection of nuclear acid,protein Ionic have made a great progress in analytical systerm of microfluidic chip,expeacially in the detection of nuclear acid.K-ras mutations are common in various cancers,especially in colorectal cancer which have a prevalence of 40-50%.So the detection of Ras gene can effectively sift colorectal cancer and predict the fatalness of metastasis.In the study,PCR-RFLP have been used as an economical and simple method,The effective and rapid electrophoresis chip associate with PCR-RFLP can rapidly detect the mutation of the gene associate with tumor.It is also more important that association of PCR,enzyme cutting and electrophoresis settles the foundation of microanalytical system.Materials and MethodsMicrochips were fabricated by using standard photolithography and wet chemical etching techniques.The etched plate and the cover plate were bonded together via fusion bonding.A home-build LIF detection device and a PMT detector were employed for detection.1.modified microchannle with acid buffer and dynamic coat of HPC2.Optimizing silting systemIn the same silting density,observe the effects of different intensive buffer and different electronic intensity:120V/cm,180V/cm,240 V/cm.observe the effects of different density of silting media such as 1.0%,2.0%,3.0%3.The microchip's detecting for colon cancer (1)cultivating cell series and collecting tissue of colon cancers.(2)establishing method of PCR-RFLP.(3)according to coden 12 of K-ras gene,establishing mixed model of mutation and wild proportion 1:1,1:10,1:100,1:10~3,1:10~4,1:10~5,1:10~6. Comparing microchip and general electrophoresis.and observing the limitation of detection on mutation;Diluting respectively the samples OF PCR-RFLP by 50,100,200,300,400.500,600 times and check their sensitivity.(4)detecting six cell series and the 46 clinical examples,observing the sensitivity and rate of match case, comparing with normal electrophoresis and check it make sense in silting process.Results1.The applicability of the coating method with MES(pH6.1)and HPC dynamic modification could suppress EOF and increase separate degree effectively.2.Optimizing system:In the same silting density,3XTBE and 180V/cm can separateφX174-HaeⅢRF DNA in better effect and relatively short time.In the same electronic intensity,2%HPC is better the separate effect as the increasing of density.The test also verify 180V/cm can have a better effect.3.The microchip's measuring for cells of colorectal cancer:(1)aimed at K-ras gene coden 12,the method of PCR-RFLP is founded.(2)Establishing mixed model of mutation and wild proportion,analysed by the microchip,the detective limitation can reach 1:10000 and its counterpart can only reach 1:10 with a blur strap;Diluting the samples respectively,the apices can be seen clearly in 500 times condition,sensitivity can reach pg or even smaller,comparing with agorose electrophroesis,the sensitivity enhances by 500 times.4.The results of detecting six cell series and the 46 clinical examples is the same as proposed sensitive 100%。Two sample appears mutation,as DNA sequencing and traditional electrophoresis have no envidence,it is quite dfferent rate between the two methods. ConclusionMicrochip with acid buffer and HPC dynamtic modification could improve separate degree and sensitive effectively.Assosated with one step PCR-RFLP,the limit of detection can be 1:10000,it is 1000 times than the traditional electrophoresisa.The relativity could reach 100%and the sensitivity is more than 500 times than normal electrophoresis,so it provides a effective analysis tool for detecting tumour genes and establishing uTAS.