The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels

Abstract

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.

The effect of genome length on ejection forces in bacteriophage lambda

“…A 10 ng quantity of a standard ladder (λ-mix, Fermentas) was included for size comparison. We ran the gel using the method of (Birren et al, 1990), with a simple electrophoresis box (Owl Separation Systems B1A) and a homebuilt voltage inverter, pulsed at 100 V forward, 60 V backward, for 19 h. The gel was stained with SYBR Gold (Molecular Probes) and photographed with an Alphaimager HP (Alpha Innotech). Dust was manually erased from the resulting image, which is shown in Fig.…”

Microscopic behaviour of DNA during electrophoresis: electrophoretic orientation

“…Also, the field strength may be modulated differently in the two directions: in the zerointegrated field electrophoresis method (ZIFE) this possibility is exploited to force DNA molecules above a certain size to become effectively stationary, which results in sharp separation (Turmel et al 1990). FIGE with asymmetric voltages has been found to improve considerably the separation of DNA fragments of 50 kbp and less (Birren et al 1990). Also the limiting case of zero field backward pulse (intermittent-field gel electrophoresis, IFGE) is known to improve the separation (Jamil et al 1989;Sutherland et al 1987) but leads to unpractically long times for sizes above 300 kbp (Lalande et al 1987), presumably as a result of slow relaxation (Lai et al 1988).…”

Field inversion gel electrophoresis in denaturing polyacrylamide gels

“…There have been few publications where inverted and intermittent fields were applied to sequencing gels (13)(14)(15) but the improvements in separation seem to be rather small and the results do not give a clear picture about the behavior of single stranded DNA molecules in pulsed field sequencing gels. Therefore we have undertaken a systematic study, to achieve a better understanding about the mobility of single stranded DNA molecules in sequencing gels under non-stationary electrophoresis conditions and to achieve optimal and reproducible separation.…”