Janning P, Schrader W, Linscheid M
ISAS Institut für Spektrochemie und Angewandte Spektroskopie, Dortmund, Germany.
Rapid Commun Mass Spectrom. 1994 Dec;8(12):1035-40. doi: 10.1002/rcm.1290081226.
A new approach is described for the enzymatic digestion of DNA yielding oligonucleotides ranging from dinucleoside monophosphates to octanucleoside heptaphosphates. DNA was digested by means of the benzon nuclease, an unspecific nuclease, and alkaline phosphatase to remove the terminal phosphate. The mixture of oligonucleotides was separated using capillary-zone electrophoresis with a buffer system, yielding a rather strong electro-osmotic flow. The oligomers are separated into groups with nucleotides of the same chain length. The separation capillary was used as the innermost capillary of an electrospray spraying system. Negative molecular ions of the nucleotides were recorded using a home-built interface and ion source for a sector-field mass spectrometer. This approach allows the facile detection of DNA modifications since they lead not only to differences in mass, but also can possess altered electrophoretic mobility. For modifying reactions which exhibit sequence specificity, the information is retained in the oligomers. Thus, reactions of DNA with electrophiles can be evaluated at different levels, since in longer chains, even complex sequence specificity may be apparent. Results from calf thymus DNA digests and preliminary experiments with DNA adducts with styrene oxide are discussed.
本文描述了一种用于DNA酶促消化的新方法，该方法可产生从二核苷单磷酸到八核苷七磷酸的寡核苷酸。DNA通过苯甲酰核酸酶（一种非特异性核酸酶）和碱性磷酸酶进行消化，以去除末端磷酸基团。使用具有缓冲系统的毛细管区带电泳分离寡核苷酸混合物，产生相当强的电渗流。寡聚物按链长相同的核苷酸分组分离。分离毛细管用作电喷雾系统的最内层毛细管。使用自制的接口和扇形场质谱仪的离子源记录核苷酸的负分子离子。这种方法能够轻松检测DNA修饰，因为它们不仅会导致质量差异，还可能具有改变的电泳迁移率。对于表现出序列特异性的修饰反应，信息保留在寡聚物中。因此，DNA与亲电试剂的反应可以在不同水平上进行评估，因为在较长的链中，甚至复杂的序列特异性也可能很明显。文中讨论了小牛胸腺DNA消化的结果以及与环氧苯乙烯形成的DNA加合物的初步实验。
