Variable Frequency Modulation in DNA Separations Using Field Inversion Capillary Gel Electrophoresis

Abstract

Variable frequency pulsed field capillary gel electrophoresis of nucleic acid restriction fragment mobility and resolution is reported. The frequency of the sine wave superimposed on the dc driving field was randomly varied throughout the duration of the electrophoresis in order to enhance the fragment separations. By frequent changes of frequency, fragments of various sizes experienced optimum conditions for separation sometime during the electrophoresis. The mobilities of phi X 174 HaeIII fragments in 9% T linear polyacrylamide were primarily dependent on power dissipation in the capillary, independent of whether the driving field was dc only or modulated. Resolution of long fragments exhibited a strong modulation frequency dependence. Modulated driving fields provided better resolution of the 603-1353 base pair fragments than dc only fields. Modulation protocols with center frequencies near 100 Hz gave the highest resolution.

Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions

“…Instead, there is a steep rising edge and a long tail. Similar band shapes have been observed in CE of ds-DNA [29] and ss-DNA [30]. Investigators rarely comment on the asymmetry or attempt to explain it.…”

Capillary Electrophoresis in Biotechnology

“…Similar to slab gels, the demonstrated limit for separations of high resolution by CE is approximately 20 000 bp (Pariat et al, 1993;Strege and Lagu, 1991), while up to 48.5 kbp have been separated with low resolution using linear PA (Guszcynski et al, 1993). Pulsed-field CE, coupled with dilute polymer solutions, has been used to separated DNA of >23 kbp (Sudor and Novotny, 1994;Navin et al, 1994;Kim and Morris, 1994) and separation of DNA as large as 1.6 Mbp using an ultradilute mixture of HEC and PEO was recently reported (Kim and Morris, 1995).…”

“…, 1993). Pulsed‐field CE, coupled with dilute polymer solutions, has been used to separated DNA of >23 kbp (Sudor and Novotny, 1994; Navin et al, 1994; Kim and Morris, 1994) and separation of DNA as large as 1.6 Mbp using an ultradilute mixture of HEC and PEO was recently reported (Kim and Morris, 1995).…”

High‐performance field inversion capillary electrophoresis of 0.1–23 kbp DNA fragments with low‐gelling, replaceable agarose gels

“…However, by bending the capillary into a figure-eight or a serpentine path, the decrease in resolution can be strongly suppressed [28, 301. Navin, Rap and Morris [31] have developed a FICGE method for the separation of small DNA fragments based on variable frequence modulation and Noolandi [32] has described a slab gel technique in which the pulse widths and amplitudes are continuously varied during the course of a separation.…”