Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).
Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).
复制标题
核酸的非放射性标记和检测。
DOI：
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
K. Mühlegger
中科院分区：
文献类型：
--
作者：
C. Kessler;H. Höltke;R. Seibl;J. Burg;K. Mühlegger
基于酶联免疫吸附法原理，建立了一种新型的高灵敏度非放射性DNA标记检测系统。通过用Klenow酶酶促掺入地高辛标记的脱氧尿苷三磷酸，用强心苷-半抗原地高辛修饰DNA。地高辛配基通过11个原子的线性间隔基（Dig-[11]-dUTP）与dUTP连接。膜结合靶DNA与地高辛标记探针杂交后，使用与标记酶碱性磷酸酶共价偶联的地高辛特异性抗体通过ELISA反应检测杂交体[（Dig）：CIAP]。抗体的这种结合：标记酶-缀合物之后是与有色底物5-溴-4-氯-3-吲哚基磷酸盐（BCIP）和硝基蓝四唑盐（NBT）的酶催化偶联氧化还原反应，产生直接粘附于膜的深蓝色的水不溶性沉淀物。地高辛系统允许在硝酸纤维素或尼龙膜上的斑点和Southern印迹中在16小时内检测0.1 pg同源DNA，即使在长时间显色后也避免任何显著的背景。由于其高灵敏度和特异性，新系统适用于检测基因组印迹中的单拷贝基因以及北方、狭缝、菌落、空斑和原位杂交。
A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.
非放射性DNA杂交探针检测人类免疫缺陷病毒核酸的比较。
影响因子：
3.3
作者：
Donovan,RM;Bush,CE;Peterson,WR;Parker,LH;Cohen,SH;Jordan,GW;VandenBrink,KM;Goldstein,E
通讯作者：
Goldstein,E
DOI：
10.1073/pnas.80.13.4045
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LEARY, JJ;BRIGATI, DJ;WARD, DC
通讯作者：
WARD, DC