Wu H, Morgan R L, Aboleneen H
Abbott Laboratories, Diagnostics Division, Abbott Park, Illinois 60064-3500, USA.
J Am Soc Mass Spectrom. 1998 Jul;9(7):660-7. doi: 10.1016/S1044-0305(98)00050-6.
A simple and powerful method for the determination of labeling sites on oligodeoxynucleotides (ODN) has been developed. The method is based on the finding that nuclease P1 (NP1) digestions of label-containing ODNs produce site-specific products: 5'-labeled ODNs produce label-nucleotide (L-N); 3'-labeled ODN produces phosphorylated label (pL); and a label in between the ODN termini produces pL-N. Mass spectrometry spectra of these products from the digestion mixture can be easily utilized for structural verification of labeled ODNs such as DNA probes. We also developed a method for the determination of the labeling sites of ODNs with unknown label structures. In this method, NP1 digestion products generate site-specific fragmentation patterns upon collision-induced dissociation. These patterns can be easily recognized and used for the identification of labeling sites of ODNs with unknown label structures. When an ODN is internally labeled, phosphodiesterase digestion may be used to determine the exact labeling site (sequence location). It was demonstrated that these methods can be applied for ODNs with single or multiple labels, and for ODNs with the same or different labels within an ODN.
已开发出一种用于确定寡脱氧核苷酸（ODN）标记位点的简单且强大的方法。该方法基于以下发现：含标记的ODN经核酸酶P1（NP1）消化会产生位点特异性产物：5'标记的ODN产生标记核苷酸（L-N）；3'标记的ODN产生磷酸化标记（pL）；而位于ODN末端之间的标记产生pL-N。消化混合物中这些产物的质谱图可轻松用于验证标记ODN（如DNA探针）的结构。我们还开发了一种用于确定标记结构未知的ODN标记位点的方法。在该方法中，NP1消化产物在碰撞诱导解离时会产生位点特异性裂解模式。这些模式可轻松识别并用于鉴定标记结构未知的ODN的标记位点。当ODN进行内部标记时，可使用磷酸二酯酶消化来确定确切的标记位点（序列位置）。结果表明，这些方法可应用于具有单个或多个标记的ODN，以及ODN内具有相同或不同标记的ODN。
