ENTRY OF DNAs INTO GELS |
161 |
Figure 5.33 How DNA knotting may prevent gel entry. Jean Viovy has obtained evidence that is consistent with the notion that knots are responsible for irreversible trapping
of DNA within the gels. But there is still no direct proof for this idea (Viovy et al., 1992).
BOX 5.2
PRACTICAL ASPECTS OF DNA ELECTROPHORESIS
The ideal PFG fractionations would be rapid, high-yield resolution separations and allow larger quantities of sample to be purified to increase the ease and sensitivity of subsequent analyses. This ideal is not achievable because both speed and sample size generally lead to a loss in resolution, for reasons that are not fully understood.
The quality of PFG separations strongly deteriorates as DNA concentration is raised. Samples should be run at the lowest concentrations that allow proper visualization or other necessary analysis of the data or utilization of the separated samples. Examples are presented in Panel A.
(continued)
162 PRINCIPLES OF DNA ELECTROPHORESIS
BOX 5.2 (Continued)
Panel A. Effect of DNA Concentration on Apparent Electrophoretic Mobility (Adapted from Doggett et al. 1992)
In ordinary agarose electrophoresis of DNA, most band broadening occurs not by dif- |
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fusion but by an electrical-field-dependent process called |
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dispersion. |
The factors that |
produce this dispersion remain to be clarified (Yarmola et al., 1996). |
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In PFG separations in all generally used apparatus, |
a |
trade-off |
must |
be |
made |
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between band sharpness (resolution) and how straight the lanes are. The use of field |
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gradients, an inherent property of the first PFG instrument designs, leads to band |
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sharpening. However, no successful design of a PFG apparatus has been demonstrated |
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that produces straight lanes and still allows gradients |
to |
be |
present |
to |
sharpen |
the |
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bands. Examples of the trade-offs before resolution and |
straightness |
are |
shown |
in |
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Panel B. |
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Panel B. Effect of a Gradient in Electrical Field on the Apparent Sharpness |
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of Bands |
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The basic mechanism by which gradients lead to band |
sharpening is |
shown |
in |
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Panel C: |
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(continued)
ENTRY OF DNAs INTO GELS |
163 |
BOX 5.2 (Continued)
Panel C. Rationale of the Effect of Electrical Field Gradients
Because molecules at the back of the zone are moving faster than those at the front, they will catch up until limits on zone thickness are reached that broaden the zone either by dispersion or by concentration-dependent effects.
Band sharpening can also be achieved by other methods. For example, a gradient of increasing gel concentration will mimic the effect of a gradient of decreasing electrical field strengths.
164 PRINCIPLES OF DNA ELECTROPHORESIS
SOURCES AND ADDITIONAL READINGS
Birren, B., and Lai, E., eds. 1993. |
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Pulsed Field Gel Electrophoresis |
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. San Diego: Academic Press. |
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Birren, B., and Lai, E. 1994. Rapid pulsed field separation of DNA molecules up to 250 kb. |
|
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|
Nucleic |
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Acids Research |
22: 5366–5370. |
|
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|
|
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|
|
|
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Bustamante, C., Vesenka, J., Tang, C. L., Rees, W., Guthold, M., and Keller, R. 1992. Circular DNA |
|
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|
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molecules imaged in air by scanning force microscopy. |
|
|
|
|
Biochemistry |
31: 22–26. |
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Cantor, C. R., and Schimmel, P. R. 1980. |
|
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Biophysical |
Chemistry |
. San |
Francisco: W. H. Freeman, |
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ch. 24. |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Carlsson, C., and Larsson, A. 1996. |
Simulations |
of |
the |
overshoot in |
the build-up of orientation |
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of long DNA during gel electrophoresis based on |
a distribution |
of oscillation |
times. |
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Electrophoresis |
17: 1425–1435. |
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Desruisseaux, C., and Slater, G. W. 1996. Pulsed-field trapping electrophoresis: A computer simula- |
|
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|
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tion study. |
Electrophoresis |
|
17: 623–632. |
|
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Doggett, N. A., Smith, C. L., and Cantor, C. R. 1992. The effect of DNA concentration on mobility |
|
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|
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in pulsed-field gel electrophoresis. |
|
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|
Nucleic Acids Research |
20: 859–864. |
|
|
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Duke, T. A. J., Austin, R. H., Cox, E. C., and Chan, S. S. 1996. Pulsed-field electrophoresis in mi- |
|
|
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crolithographic arrays. |
Electrophoresis |
|
|
17: 1075–1079. |
|
|
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|
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Frank-Kamenetskii, M. F. 1997. |
|
|
Unraveling DNA. |
|
Reading MA: Addison-Wesley. |
|
|
|
||||||||
Gurrieri, S., Smith, S. B., Wells, K. S., Johnson, I. D., and Bustamante, C. 1996. Real-time imaging |
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of the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees and 120 |
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degrees pulsed field gel electrophoresis. |
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Nucleic Acids Research |
24: 4759–4767. |
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Monaco, A. P., ed. 1995. |
|
Pulsed |
Field |
Gel |
Electrophoresis: |
A Practical Approach |
|
|
. New York: |
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Oxford University Press. |
|
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|
|
|
|
|
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|
|
|
|
|
|
||
Nordén, |
B., Elvingson, C., |
Jonsson, |
M. and |
Åkerman, |
B. |
1991. |
Microscopic |
behavior |
of |
|
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DNA during electrophoresis: electrophoretic orientation. |
|
|
|
Quarterly Review of Biophysics |
24: |
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103–164. |
|
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Schwartz, D., and Cantor, C. R. 1984. Separation of yeast chromosome-sized DNAs by pulsed field |
|
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|
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gradient gel electrophoresis. |
|
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Cell 37: 67–75. |
|
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Smith, M. A., and Bar-Yam, Y. 1993. Cellular automaton simulation of pulsed field gel elec- |
|
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trophoresis. |
Electrophoresis |
|
14: 337–343. |
|
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|
|
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Turmel, C., Brassard, E., Slater, G. W., and Noolandi, J. 1990. Molecular detrapping and band nar- |
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rowing with high frequency modulation of pulsed field electrophoresis. |
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Nucleic Acids Research |
|
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18: 569–575. |
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Ulanovsky, L., Drouin, G., and Gilbert, W. 1990. DNA trapping electrophoresis. |
|
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Nature |
343: |
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190–192. |
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Viovy, J. L., Miomandre, F., Miquel, |
M. C., Caron, F., and Sor, |
F. 1992. |
Irreversible trapping of |
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DNA during crossed-field gel electrophoresis. |
|
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Electrophoresis |
13: 1–6. |
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Whitcomb, R. W., and Holzwarth, G. 1990. On the movement and alignment of DNA during 120 |
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degrees pulsed-field gel electrophoresis. |
|
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|
Nucleic Acids Research |
18: 6331–6338. |
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Yarmola, E., Sokoloff, H., and Chrambach, A. 1996. The relative contributions of dispersion and |
|
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|
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diffusion to band spreading (resolution) in gel electrophoresis. |
|
|
Electrophoresis |
17: 1416–1419. |
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Zhang, T.-Y., Smith, C. L., |
and Cantor, |
C. R. |
1991. |
Secondary pulsed field gel electrophoresis: |
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a |
new method |
for faster separation |
of |
larger |
DNA |
molecules. |
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Nucleic |
Acids Research |
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19: 1291–1296.