Study On The Separation And Detection Of DNA Fragments With A Wide Molecular Weight Range By Capillary Gel Electrophoresis And Non-gel Capillary Electrophoresis

The rapid development of the Human Genome Project has promoted the application of technologies for the nucleic acid analysis in mutation detection,molecular diagnosis,disease prevention,and treatment evaluation.It is quite important to develop methods with fast and easy quantification,automation,and high-throughput.Capillary electrophoresis(CE)has the advantages of high separation efficiency,many operating modes,and low sample consumption.It has become an indispensable tool for the separation and analysis of biological macromolecules such as proteins and nucleic acids.capillary gel electrophoresis(CGE)and non-gel capillary electrophoresis(NGCE)are common used.CGE effectively separates small molecular weight DNA fragments ranging from 50 to2000 bp with extremely high resolution.However,the large viscosity of the gel,it is easy to produce bubbles in the process of preparation of CGE capillary colums,resulting in poor stability and reproducibility,and wider molecular weight DNA fragments,the resolution is poor.NGCE takes low-viscosity linear polymer solution as the screening medium,and the polymer in the solution interwinds each other to form mesh,so as to achieve better separation.These low viscosity polymers provide faster,more stable separation,allow greater flexibility and pressure injection,and are easier to automate.NGCE can also provide larger pore for the separation of DNA fragments with a wider molecular weight range,which can improve the reading length of large molecular fragments by DNA sequencing.In this paper,CGE and NGCE were used to separate and analyze DNA fragments with a wide molecular weight range.The methodology was evaluated.The problems of the method were found,and a new method was also established to prepare LPA coated capillary column to improve the reproducibility and service life.The main contents of this thesis are as follows:Chapter 1,the significance of nucleic acid analysis and common technical methods are reviewed.The basic concepts of CGE and NGCE and their applications in nucleic acid analysis are introduced.Chapter 2,a linear polyacrylamide(LPA)coated capillary column made by literature methods was used as the separation channel,and LPA was used as the gel of CGE.A method for the separation of DNA fragments in the molecular weight range of 100 ~ 2,000 bp was established.Under the optimized CGE separation conditions,QDL 2,000 DNA Marker(including six DNA fragments of 100 bp,250 bp,500 bp,750 bp,1000 bp,and 2000 bp)was selected as the test sample,and the functional relationship between the molecular weights and migration times of the DNA fragments were established.The methodology was validated and applied to the qualitative and quantitative analysis of DNA fragments in PCR amplified products.The results showed although this method solved the bubble problem when preparing a gel column,the CGE gel column had a short service life and required multiple gel replacements.This meight be due to the unstability gel of CGE.Chapter 3,an NGCE method for the separation of DNA fragments in the molecular weight range of 250 ~ 10000 bp was established,by using an LPA coated capillary column made by literature methods as the separation channel,and LPA as the screening medium.Under optimized separation conditions,the methodological validation was carried out.DL10,000 DNA Marker(including seven DNA fragments of 250 bp,500 bp,1000 bp,2000 bp,4000 bp,7000 bp,and 10,000 bp)were selected as the test samples.The results showed that it was accurate,reliable and reproducible for DNA fragments with molecular weights below2000 bp;it was poor for DNA fragments with molecular weights above 2000 bp.This meight be due to the poor stability of the coated capillary column.The coating was easy to fall off,resulting in the adsorption of large molecular weight DNA fragments(more than 2000 bp)on the inner wall of the capillary column.Chapter 4,LPA-coated capillary columns were prepared by in-situ polymerization in the pretreatment capillaries with acrylamide(AA),ammonium persulfate(APS),and N,N,N',N'-tetramethylethylenediamine(TEMED)as the reaction monomer,initiator,and accelerator,respectively.Different concentrations of AA in the prepolymerization solutions were employed to optimize the preparation of LPA coated capillary colums.The DL 10,000 DNA Marker was selected as the test sample.Under the optimized NGCE conditions inChapter 3,the separation performance of LPA-coated capillary columns for DNA fragments in the molecular weight range of 250 ~ 10,000 bp was investigated.In-column and inter-column stability were evaluated.The results showed that the LPA-coated capillary column coming from 5% AA had the best stability and longest service life.It could be used for the separation and analysis of DNA fragments in a wide molecular weight range.Chapter 5,conclusions and prospects.