Electrophoresis of DNA in Oriented Agarose Gels

Holmes, Diana L.; Stellwagen, Nancy C.

doi:10.1080/07391102.1989.10507774

Cited by 20 publications

(16 citation statements)

References 14 publications

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“…In analogy with the results described here, electrical fieldinduced orientation of a either molten [29] or gelled [30,31] agarose sieving matrix has previously been observed. The effect on the molten agarose was observed via subsequent gelation and electrophoretic sieving [29].…”

Section: Possible Mechanism Of Preconditioningsupporting

confidence: 71%

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Matrix conditioning for lengthened capillary DNA sequencing

2005

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Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.

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Section: Possible Mechanism Of Preconditioningsupporting

confidence: 71%

“…The effect on the molten agarose was observed via subsequent gelation and electrophoretic sieving [29]. Experimental tests have apparently also not yet rigorously demonstrated the cause for the field-induced effects on agarose sieving matrices.…”

Section: Possible Mechanism Of Preconditioningmentioning

confidence: 97%

Matrix conditioning for lengthened capillary DNA sequencing

2005

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“…The discordant agarose and polyacrylamide gel results are most likely due to the fact that the agarose gel fibers, which aggregate into large fiber bundles held together by hydrogen bonds [35], can be oriented by electric fields of the amplitudes typically used for agarose gel electrophoresis [35][36][37][38][39]. The orientation of the agarose fibers and fiber bundles effectively creates transient pores or "tunnels" through the gel matrix in the direction of the electric field [36]. Small mobility differences between curved and normal DNA molecules containing the same number of bp would not be observable under such conditions, especially since the bands observed for DNA in agarose gels are relatively broad.…”

Section: Discussionmentioning

confidence: 99%

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

Stellwagen

2006

Electrophoresis

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The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.

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“…The DNA mobilities and trajectories return to normal if the preoriented gel is allowed to stand a t room temperature for -24 h before electrophoresis is started, suggesting that the structure of the gel becomes randomized upon standing. 74 The results clearly suggest that the orientation of agarose fibers and domains can affect the electrophoretic mobilities of DNA fragments. In addition, structural rearrangements of the agarose matrix require very little external energy.…”

Section: Revelance To Pfgementioning

confidence: 95%

Transient electric birefringence of agarose gels. I. Unidirectional electric fields

Stellwagen

1994

Biopolymers

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The orientation of agarose gels in pulsed electric fields has been studied by the technique of transient electric birefringence. The unidirectional electric fields ranged from 2 to 20 V/cm in amplitude and 1 to 100 s in duration, values within the range typically used for pulsed field gel electrophoresis (PFGE). Agarose gels varying in concentration from 0.3 to 2.0% agarose were studied. The sign of the birefringence varied randomly from one gel to another, as described previously [J. Stellwagen & N.C. Stellwagen (1989), Nucleic Acids Research, Vol. 17, 1537-1548]. The sign and amplitude of the birefringence also varied randomly at different locations within each gel, indicating that agarose gels contain multiple subdomains that orient independently in the electric field. Three or four relaxation times of alternating sign were observed during the decay of the birefringence. The various relaxation times, which range from 1 to approximately 120 s, can be attributed to hierarchies of aggregates that orient in different directions in the applied electric field. The orienting domains range up to approximately 22 microns in size, depending on the pulsing conditions. The absolute amplitude of the birefringence of the agarose gels increased approximately as the square of the electric field strength. The measured Kerr constants are approximately 5 orders of magnitude larger than those observed when short, high-voltage pulses are applied to agarose gels. The increase in the Kerr constants in the low-voltage regime parallels the increase in the relaxation times in low-voltage electric fields. Birefringence saturation curves in both the low- and high-voltage regimes can be fitted by theoretical curves for permanent dipole orientation. The apparent permanent dipole moment increases approximately as the 1.6 power of fiber length, consistent with the presence of overlapping agarose helices in the large fiber bundles orienting in low-voltage electric fields. The optical factor is approximately independent of fiber length. Therefore, the marked increase in the Kerr constants observed in the low-voltage regime is due to the large increase in the electrical orientation factor, which is due in turn to the increased length of the fiber bundles and domains orienting in low-voltage electric fields. Since the size of the fiber bundles and domains approximates the size of the DNA molecules being separated by PFGE, the orientation of the agarose matrix in the applied electric field may facilitate the migration of large DNA molecules during PFGE.

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Electrophoresis of DNA in Oriented Agarose Gels

Cited by 20 publications

References 14 publications

Matrix conditioning for lengthened capillary DNA sequencing

Matrix conditioning for lengthened capillary DNA sequencing

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

Transient electric birefringence of agarose gels. I. Unidirectional electric fields

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