Effects of Linear Polyacrylamide Concentrations and Applied Voltages on the Separation of Oligonucleotides and DNA Sequencing Fragments by Capillary Electrophoresis

• Published in: Analytical chemistry (Washington) Vol. 66; no. 23; pp. 4243 - 4252
• Main Authors: Manabe, Takashi, Chen, Nong, Terabe, Shigeru, Yohda, Masafumi, Endo, Isao
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.12.1994
• Subjects: Acrylic Resins - chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Cross-Linking Reagents - chemistry; Deoxyribonucleic acid; DNA; DNA - chemistry; Dna, deoxyribonucleoproteins; Electrophoresis, Polyacrylamide Gel; Fluorescein-5-isothiocyanate - chemistry; Fundamental and applied biological sciences. Psychology; Instruments; Nucleic acids; Oligonucleotides - isolation & purification; Reproducibility of Results; Spectrometry, Fluorescence; Performance evaluation; Separation method; Nucleotide sequence; Gel electrophoresis; DNA; Acrylamide polymer; Optimization; Capillary tube; Structural analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac00095a020

Oligonucleotides and DNA sequencing fragments have been separated by capillary electrophoresis employing linear polyacrylamide (LPA) as a sieving matrix. A commercially available apparatus equipped with a laser-induced fluorescence (LIF) detection system has been utilized, but the capillary cartridge has been modified to position the capillaries without coiling. The performance of the separation, the relationship between resolution and analysis time, has been examined using poly(dT)16-500 by changing LPA concentration, capillary length, and electric field strength. It was found that, for large DNA fragments, the migration time interval between bands decreases linearly as DNA fragment size increases. This implies that there exists a maximum base number to be resolved, irrespective of the band width (we named the maximum base number Nmax). The higher value of Nmax is obtained when the applied field strength is lower, but this accompanies longer analysis time with a concomitant increase in band width. Simple experimental equations have been proposed to calculate resolution and migration times of DNA fragments separated in our system at given electrophoretic conditions. Using 9% T LPA and an electric field strength of 100 V/cm, single-base resolution of M13mp10 DNA fragments up to 520 nucleotides has been obtained.