Urdea M S, Warner B D, Running J A, Stempien M, Clyne J, Horn T
Chiron Corporation, Emeryville, CA 94608.
Nucleic Acids Res. 1988 Jun 10;16(11):4937-56. doi: 10.1093/nar/16.11.4937.
N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.
已合成了N4-[N-(6-三氟乙酰氨基己酰基)-2-氨基乙基]-5'-O-二甲氧基三苯甲基-5-甲基-2'-脱氧胞苷-3'-N,N-二异丙基-甲基亚磷酰胺。这种N4-烷基氨基脱氧胞苷衍生物已在化学DNA合成过程中被掺入寡核苷酸探针中。在脱保护和纯化之后，已特异性地掺入了荧光（荧光素、德克萨斯红和罗丹明）、化学发光（异鲁米诺）和酶（辣根过氧化物酶、碱性磷酸酶）标记物。评估了标记物和标记探针的检测限。此外，还测定了夹心杂交试验中标记探针的检测限和非特异性结合。发现酶修饰的寡核苷酸是比荧光或化学发光衍生物显著更好的标记材料，其灵敏度与32P标记的探针相当。
