DNA Mutation Detection Platform And Approache Based On Digital Microfluidic

With the deepening of human genome research,DN A mutation analysis is of great importance in many areas,including studies of gene function,discovery of disease-related-marker,and clinical diagnosis.As report,tumors are caused by the instability of human genome and the mutation of related genes.Therefore,the detection of gene mutation is of great significance in the early diagnosis of tumors.Traditional detection approaches for gene mutations,including allele specific amplification,high resolution melting,and DNA sequencing.These were limited by their low sensitivity,poor robustness and harmful method,which can’t meet the current needs of genetic mutation detection.In addition,the sequencing methods are not well suited for community hospitals,clinics and laboratories due to the high instrumentation costs and complex procedures.As a result,a simple,low-cost,automated,sensitive mutation detection system is highly desirable.Digital microfluidics is an emerging technique to handle discrete picoliter to microliter scale droplets.As a new kind of microfluidic technology,it keeps the traditional microfluidic less sample consumption,fast heat conversion,automation and flexibility.Beside,due to that it do not rely on micro pump,valve or mixer and other components,it is suitable analysis system with high integration,complex operation for micro biochemical sequencing.In this thesis,we have developed a Palmtop Pyrosequencer based on the pyrosequencing principle and implemented by digital microfluidics.All pyrosequencing reaction steps were completed on DMF chip by fully automatic microdroplet manipulation,realizing real-time controllable and automatic reading of sequencing results.By analyzing single nucleotide polymorphisms(SNP)on this platform,the analysis of short sequence single nucleotide polymorphisms can be achieved within 30 min.Mutations in K-ras can be detected in clinical samples within half an hour using the Palmtop Pyrosequencer.With the Palmtop Pyrosequencer for methylation analysis,found that the sensitivity is good,and it can detect the level of 10%,and the sensitivity can be as low as 0.57 fmol,through the analysis in human genome-wide adding samples,different levels of methylation can be found.It has practical application in methylation detection.In addition,we also developed a fully automated single-cell capture based on digital microfluidic,which successfully achieved 100%single-cell capture within 1 min,The detection of EGFR mutation on single-cell level has been successfully achieved by using time-of-flight mass spectrometry,which is expected to further promote the routine testing of efficient and accurate tumor-targeted therapy.