Turmel C, Noolandi J
Xerox Research Centre, Mississauga, Ontario, Canada.
Electrophoresis. 1993 Apr;14(4):304-12. doi: 10.1002/elps.1150140153.
A summary of the three main one-dimensional pulsed-field strategies (zero-integrated field, forward-biased field, and high frequency modulation) used for separating DNA molecules without band inversion within a preselected size range is given. Each of these strategies has size-specific features which make separations up to 6 Mbp possible. We applied the same methodology to circular DNAs varying in size from 2 kbp to about 4 Mbp. The migration of intermediate-sized circular plasmids (50 kbp-400 kbp) under these pulse conditions remains unexplained. On the other hand, preliminary results show that the migration of very large molecules, which are expected to be circular, comigrate with linear chromosomes of the same size under certain pulse conditions. We hypothesize that sample preparation, or the effect of the pulsed field, can create breakage and linearize very large circular DNAs, or that very large circular DNAs (> 2 Mbp) act like linear DNAs of the same size when submitted to one-dimensional pulsed-field gel electrophoresis conditions. The most likely possibility is that some of the circular DNAs have been linearized with one break during sample preparation, giving rise to a band at about 4 Mbp. The circular DNAs with more than one break may form an indistinguishable smear.
本文给出了三种主要的一维脉冲场策略（零积分场、正向偏置场和高频调制）的总结，这些策略用于在预选大小范围内分离DNA分子而不发生条带反转。每种策略都有特定大小的特征，使得分离长达6 Mbp的DNA成为可能。我们将相同的方法应用于大小从2 kbp到约4 Mbp不等的环状DNA。在这些脉冲条件下，中等大小的环状质粒（50 kbp - 400 kbp）的迁移情况仍无法解释。另一方面，初步结果表明，在某些脉冲条件下，预期为环状的非常大的分子的迁移与相同大小的线性染色体共迁移。我们假设样品制备或脉冲场的作用会导致非常大的环状DNA断裂并线性化，或者在进行一维脉冲场凝胶电泳条件时，非常大的环状DNA（> 2 Mbp）的行为类似于相同大小的线性DNA。最有可能的情况是，一些环状DNA在样品制备过程中发生了一次断裂而线性化，从而在约4 Mbp处产生一条带。具有多个断裂的环状DNA可能会形成难以区分的拖尾。
