ORIENTATIONAL DYNAMICS OF T2 DNA DURING AGAROSE-GEL ELECTROPHORESIS - INFLUENCE OF GEL CONCENTRATION AND ELECTRIC-FIELD STRENGTH
ORIENTATIONAL DYNAMICS OF T2 DNA DURING AGAROSE-GEL ELECTROPHORESIS - INFLUENCE OF GEL CONCENTRATION AND ELECTRIC-FIELD STRENGTH
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DOI：
10.1002/bip.360280906
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发表时间：
1989-09-01
期刊：
影响因子：
2.9
通讯作者：
LALANDE, M
中科院分区：
文献类型：
--
作者：
AKERMAN, B;JONSSON, M;LALANDE, M
在分子水平上理解当使用调制电场时负责DNA凝胶电泳中的改进的分离的机制需要关于DNA/凝胶系统中的构象分布和动力学的详细信息。由于电泳迁移（“电泳取向”）而产生的取向顺序是本文中的一条有趣的信息，其可以通过线性二色性光谱获得[M. Jonsson，B. Akerman和B。Norden，（1988）Biopolymers 27，381-414]。该技术允许在电泳期间测量凝胶中电泳区内DNA的取向因子S（S = 1对应于完美取向）。据报道，T2 DNA [170千碱基对（kpb）]的取向程度是相当大的（在10 V/cm下在1%琼脂糖中S = 0.17），与较早发现的短片段的相对适度的取向（对于23-kbp DNA，在10 V/cm下在1%琼脂糖中S = 0.03）相比，这表明大的DNA螺旋在迁移期间基本上变形。还报道了T2 DNA的取向顺序的生长和弛豫动力学，作为凝胶浓度（0.3-2%）、电场强度（0-40 V/cm）和脉冲特性的函数。当施加恒定场时，DNA取向的上升曲线是一个非单调函数，在其达到稳态取向之前（在1%琼脂糖中12 s后，9 V/cm）显示出明显的过冲，随后是轻微的下冲。在没有磁场的情况下，取向弛豫在大约10 s的时间区域内表现出多指数衰减，此时大部分DNA各向异性已经消失。令人惊讶的现象是DNA/凝胶系统对先前脉冲的几分钟的记忆：对于两个连续的矩形脉冲（具有相同极性），与第一脉冲相比，作为对第二脉冲的响应的取向过冲和下冲显著减少。恢复90%的振幅所需的时间通常为1200 s（1%琼脂糖，9 v/cm），这可以与松弛90%的DNA取向所需的时间（仅为6 s）进行比较。因此，超调和欠调恢复的主要部分是DNA已经随机定向的系统的重组。不同的响应幅度和弛豫时间，包括过冲的幅度和恢复时间，在电泳凝胶中的DNA的取向顺序的凝胶浓度和场强的函数进行了研究。结合高分子动力学的相关理论对结果进行了讨论。
The understanding, on a molecular level, of the mechanism responsible fo r the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration ("electrophoretic orientation") is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Akerman, and B. Norden, (1988) Biopolymers 27, 381-414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (s = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23-kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3-2%), electric field strength (0-40 V/cm), and pulse characteristics. The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady-state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 v/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over- and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented. The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.