Production of polyacrylamide gel filled capillaries for capillary gel electrophoresis (CGE): Influence of capillary surface pretreatment on performance and stability

Self Cite

Abstract. Diffusion coefficients of oligonucleotides have been measured in polyacrylamide gel capillaries without application of an electrical field. A relationship between such diffusion coefficients and molecular size of the oligonucleotides could be achieved. The slower diffusion of larger oligonucleotides cannot explain the high separation efficiencies of capillary gel electrophoretic separations alone. By theoretical considerations it became obvious that diffusion coefficients of oligonucleotide molecules and also their intermolecular interaction with the polyacrylamide gel may be considerably changed by application of electrical fields.

Section: Resultsmentioning

confidence: 99%

Diffusion coefficients of oligonucleotides in capillary gel electrophoresis

Yin

Kleemiss

Lux

et al. 1991

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“…Capillaries filled with non-crosslinked polyacry1am.de. Capillary columns filled with noncrosslinked polyacrylamide were prepared as follows [9,13]. Fused-silica capillary tubing (375 pm o.d., 75 pm i.d., Polymicro Technologies, Phoenix, AZ, USA) was cut into 40-to 115-cm segments with detection windows located 11 cm from one end.…”

Section: Experimextalmentioning

confidence: 99%

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

Chen

Manabe

Terabe

et al. 1994

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.

“…Up to 20% polyethylene glycol was added to the acrylamide polymerization mixture, and a bifunctional reagent covalently bound the gel to the capillary wall [36,37]. Yin et al claimed that polyacrylamide covalently bound to the capillary wall prevented the bubble formation [38]. Holloway used a polydimethylsiloxane coupling layer, explaining that gel shrinkage was followed by polyorganosilane stretching which allows the formation of gel capillaries without voids [39].…”

Section: Separation Matrtxmentioning

confidence: 99%

Capillary gel electrophoresis

Dolnı́k

1994

Capillary gel electrophoresis (CGE) is a new rapidly developing method for biopolymer separations. It has been used successfully for separations of oligonucleotides, oligosaccharides, polyamino acids, and even for SDS electrophoresis of polypeptides. Shrinkage of the gel during polymerization, with resulting void formation as the main technological problem when making capillary gels, was overcome by several procedures. More recently, a modification of gel electrophoresis, using replaceable linear polymer solutions has gained popularity, especially for SDS electrophoresis of proteins and electrophoresis of DNA. THEORY OF GEL ELECTROPHORESISThe behavior of a charged molecule in an electric field is described in many books and papers Ell-141. All of these theoretical treatments used for electrophoresis in free solution are also valid in sieving matrices. In gel electrophoresis, the interaction between solute and matrix has the primary effect on the migration behavior. The theory of gel electrophoresis was developed primarily for classical gel electrophoresis; however, it can be applied to the capillary format as well. The expression of separation efficiency of classical gel electrophoresis