Jablonski E, Moomaw E W, Tullis R H, Ruth J L
Nucleic Acids Res. 1986 Aug 11;14(15):6115-28. doi: 10.1093/nar/14.15.6115.
Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.
使用同双功能试剂辛二酸二琥珀酰亚胺酯，已将短的合成寡核苷酸与碱性磷酸酶共价交联。这些寡聚物长度为21至26个碱基，与单纯疱疹病毒、乙型肝炎病毒、空肠弯曲菌和产肠毒素大肠杆菌中发现的独特序列互补。每个寡聚物含有一个单一的修饰碱基，带有一个12原子的“连接臂”，末端为反应性伯胺。通过该胺进行交联产生的寡聚物-酶缀合物，每个酶分子由一个寡聚物组成，具有完整的碱性磷酸酶活性，并且能够在15分钟内与固定在硝酸纤维素上的靶DNA杂交。通过染料沉淀测定法直接检测杂交体，在4小时的显影时间内，对靶DNA的检测灵敏度为10(6)个分子(2×10(-18)摩尔)。该酶对寡核苷酸杂交的选择性或动力学没有明显影响，并且缀合物可以以常规方式进行杂交和解链。
