RAPID AND SENSITIVE COLORIMETRIC METHOD FOR VISUALIZING BIOTIN-LABELED DNA PROBES HYBRIDIZED TO DNA OR RNA IMMOBILIZED ON NITROCELLULOSE - BIO-BLOTS
RAPID AND SENSITIVE COLORIMETRIC METHOD FOR VISUALIZING BIOTIN-LABELED DNA PROBES HYBRIDIZED TO DNA OR RNA IMMOBILIZED ON NITROCELLULOSE - BIO-BLOTS
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DOI：
10.1073/pnas.80.13.4045
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
WARD, DC
中科院分区：
文献类型：
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作者：
LEARY, JJ;BRIGATI, DJ;WARD, DC
在生物素化的TTP类似物存在下通过切口转移制备的生物素标记的DNA探针与固定在硝酸纤维素滤膜上的DNA或RNA杂交。去除残留探针后，将滤膜与用抗生物素蛋白-DH（或链霉抗生物素蛋白）和[小牛]肠碱性磷酸酶的生物素化聚合物制备的预形成复合物一起孵育2-5分钟。然后将滤膜与5-溴-4-氯-3-吲哚基磷酸盐和硝基蓝四唑的混合物一起孵育，导致紫色沉淀物沉积在杂交位点。该程序将在1小时或更短的酶孵育期后检测1-10 pg范围内的靶序列。如果需要，孵育期可以延长至24小时，以增加杂交信号的颜色强度。在高探针浓度（250-750 ng/ml）下，生物素标记的DNA与硝酸纤维素的非特异性结合低于放射性标记的DNA，因此分析独特哺乳动物基因序列所需的杂交时间可缩短至1-2 h。这种非放射性的探针检测方法应该是通用的遗传研究使用Southern，RNA或斑点杂交协议。
Biotin-labeled DNA probes, prepared by nick-trnaslation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2-5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of [calf] intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1-10 pg range after enzyme incubation periods of 1 h or less. The incubation period can be extended up to 24 h, if required, to increase the color intensity of the hybridization signal. At high probe concentrations (250-750 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1-2 h. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA or dot-blot hybridization protocols.