Strutz K, Stellwagen N C
Department of Biochemistry, University of Iowa, Iowa City, USA.
Electrophoresis. 1998 May;19(5):635-42. doi: 10.1002/elps.1150190504.
The electrophoresis of small DNA fragments has been measured in dilute agarose and polyacrylamide gels cast and run in Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE) buffers. Ferguson plots were constructed to extrapolate the mobilities to zero gel concentration and estimate the free solution mobility of DNA. In polyacrylamide gels, in both TAE and TBE buffers, the extrapolated mobilities at zero gel concentration increased gradually with decreasing DNA molecular weight, went through a maximum at approximately 60 bp, and then decreased again. The increase in the extrapolated mobilities with decreasing molecular weight observed for DNA fragments > or = 60 bp can be attributed to transient interactions between the migrating DNA molecules and the polyacrylamide gel fibers. If such interactions are eliminated by extrapolating the mobilities to both zero gel concentration and zero DNA molecular weight, the apparent free solution mobility of DNA is found to be 3.1 x 10(-4) cm2 V(-1) s(-1) in TAE buffer and 4.2 x 10(-4) cm2 V(-1) s(-1) in TBE buffer at 20 degrees C, reasonably close to the actual free solution mobilities measured in the same two buffers by capillary electrophoresis (N. C. Stellwagen et al., Biopolymers 1997, 42, 687-703). The significantly larger electrophoretic mobility observed in TBE buffer is most likely due to the formation of nonspecific, highly charged deoxyribose-borate complexes in this buffer medium. For DNA molecules < or = 60 bp in size, the decrease in the extrapolated mobilities with decreasing molecular weight parallels the decrease in their free solution mobilities observed by capillary electrophoresis. In agarose gels, the extrapolated mobilities of small DNA molecules at zero gel concentration appear to be independent of molecular weight. The apparent free solution mobilities are found to be (3.0 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) in TAE buffer and (3.2 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) in TBE buffer. The very similar mobilities observed in the two buffer media suggest that the borate ions in TBE buffer primarily form complexes with the galactose residues in the agarose gel fibers, rather than with the migrating DNA molecules, because of mass action effects. The formation of borate-agarose complexes, increasing the net negative charge of the agarose gel fibers, appears to be responsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer (N. C. Stellwagen, Electrophoresis 1992, 13, 601-603).
已在以Tris - 乙酸 - 乙二胺四乙酸（TAE）和Tris - 硼酸 - 乙二胺四乙酸（TBE）缓冲液灌制并运行的稀琼脂糖凝胶和聚丙烯酰胺凝胶中测量了小DNA片段的电泳情况。构建了弗格森图以将迁移率外推至零凝胶浓度，并估算DNA在自由溶液中的迁移率。在聚丙烯酰胺凝胶中，无论是在TAE还是TBE缓冲液中，零凝胶浓度下的外推迁移率都随着DNA分子量的降低而逐渐增加，在约60 bp处达到最大值，然后再次下降。对于大小大于或等于60 bp的DNA片段，观察到的外推迁移率随分子量降低而增加可归因于迁移的DNA分子与聚丙烯酰胺凝胶纤维之间的瞬时相互作用。如果通过将迁移率外推至零凝胶浓度和零DNA分子量来消除这种相互作用，则发现在20℃时，DNA在TAE缓冲液中的表观自由溶液迁移率为3.1×10⁻⁴ cm² V⁻¹ s⁻¹，在TBE缓冲液中为4.2×10⁻⁴ cm² V⁻¹ s⁻¹，这与通过毛细管电泳在相同两种缓冲液中测得的实际自由溶液迁移率相当接近（N.C. Stellwagen等人，《生物聚合物》1997年，42卷，687 - 703页）。在TBE缓冲液中观察到的明显更大的电泳迁移率很可能是由于在这种缓冲介质中形成了非特异性的、高电荷的脱氧核糖 - 硼酸盐复合物。对于大小小于或等于60 bp的DNA分子，外推迁移率随分子量降低而降低与通过毛细管电泳观察到的其在自由溶液中的迁移率降低情况相似。在琼脂糖凝胶中，零凝胶浓度下小DNA分子的外推迁移率似乎与分子量无关。发现在TAE缓冲液中的表观自由溶液迁移率为（3.0±0.1）×10⁻⁴ cm² V⁻¹ s⁻¹，在TBE缓冲液中为（3.2±0.1）×10⁻⁴ cm² V⁻¹ s⁻¹。在两种缓冲介质中观察到的非常相似的迁移率表明，由于质量作用效应，TBE缓冲液中的硼酸根离子主要与琼脂糖凝胶纤维中的半乳糖残基形成复合物，而不是与迁移的DNA分子形成复合物。硼酸 - 琼脂糖复合物的形成增加了琼脂糖凝胶纤维的净负电荷，这似乎是在以TBE缓冲液灌制并运行的琼脂糖凝胶中观察到的明显增加的电渗流的原因（N.C. Stellwagen，《电泳》1992年，13卷，601 - 603页）。
