Analytical Sciences

In Situ Fluorescence Microscopic Investigation into the Dependence of Conformation and Electrophoretic Velocity of Single DNA Molecules on Acid or Spermidine Concentration

Shohei INOSHITA, Satoshi TSUKAHARA, and Terufumi FUJIWARA

Department of Chemistry, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan

A measurement cell for DNA migration was fabricated by agarose gel on a glass plate for the purpose of in situ fluorescence microscopic measurements of the electrophoretic behaviors of single giant DNA (T4GT7DNA, 165600 base pairs) molecules. The cell was connected to an original circuit that can supply a constant electric field strength to a DNA-migration-space of the cell. Conformation and electrophoretic velocity of single DNA molecules were measured at the same time as a function of acid or spermidinium concentration. As a result, the conformational change from the random-coiled to globular states and reduced electrophoretic velocities were induced by an addition of acid or spermidinium. The change of the electrophoretic velocity was quantitatively attributable to the partial neutralization of anionic phosphate groups of DNA molecules by H⁺ or tricationic spermidinium. The acid-dissociation constant of the conjugate acid of phosphate group of the DNA molecules (pK_a) and the association constant of the phosphate groups of DNA molecule with tricationic spermidinium (K_ass) were determined to be 2.0 and 5.5 × 10³ dm³ mol^-1, respectively.

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