High‐resolution separation of oligonucleotides and DNA sequencing reaction products by capillary electrophoresis with linear polyacrylamide and laser‐induced fluorescence detection

Abstract

Abstract. By employing linear polyacrylamide (LPA) as a sieving matrix, oligonucleotides and DNA sequencing fragments have been separated by capillary electrophoresis. A commercially available apparatus equipped with a laser-induced fluorescence (LIF) detection system has been utilized, but the capillary cartridge has been modified in order to set the capillaries without coiling. Single-base resolution was achieved for a mixture of FITC-labeled oligonucleotides [FITC-p(dT)] up to 500 base-long chains. Because of high resolution, minor components of FITC-p(dT) have also been separated. The mobility differences between single-base adjacent peaks of FITC-labeled DNA sequencing fragments have been detected up to 520 base-long fragments. These results confirmed that linear polyacrylamide is an excellent sieving matrix in DNA sequencing by capillary electrophoresis. Key words: capillary electrophoresis, DNA sequencing, linear polyacrylamide, oligonucleotides, gel electrophoresis INTRODUCTIONWith molecular biology and genetic engineering entering the industrial stage, the characterization of oligonucleotides and DNA sequencing products is becoming an important issue for biologists and analytical chemists. DNA sequencing technology has been constrained by the low efficiency of slab gel electrophoresis caused by complicated manual procedures and by long analysis times. To transcend this limitation, high-performance capillary gel electrophoresis (CGE) with greater separation efficiencies appears to be an attractive alternative [1][2][3][4][5][6][7][8][9][10]. However, the problems related to the instability of gel-filled capillaries in analyzing DNA sequencing reaction products hinder the fully automated operation of CGE for DNA sequencing.

Fast DNA sequencing up to 1000 bases by capillary electrophoresis using poly(N,N-dimethylacrylamide) as a separation medium

“…A number of polymers such as linear polyacrylamide (LPA) [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26], hydroxyethylcellulose (HEC) [27], polyethylene oxide (PEO) [28,29], a copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (PDMA-co-PEA) [30], fluorocarbon end-capped polyethylene glycols [31], poly-N,Ndimethylacrylamide (PDMA) [9,[32][33][34][35], and polyvinyl pyrrolidone (PVP) [36], have been used for this purpose. Among them, LPA is the most popular, having been studied extensively and shown to be a successful separation medium.…”