Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.
Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.
复制标题
使用标记有螯合基团取代补骨脂素的 DNA 探针进行灵敏的非放射性斑点杂交，并通过铕离子荧光进行定量检测。
DOI：
10.1093/nar/16.3.1181
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发表时间：
1988
影响因子：
14.9
通讯作者：
G. Valet
中科院分区：
文献类型：
--
作者：
A. Oser;W. Roth;G. Valet
描述了一种用于杂交试验的克隆DNA探针的新标记方法。重组质粒DNA插入部分单链，用限制性内切酶酶切进行标记反应，然后用控制的核酸外切酶III孵育。含硫醇的补骨脂素衍生物通过紫外线照射与质粒DNA的剩余双链区域共价结合。补骨脂素- sh基团用大量金属螯合剂（二乙基三胺五乙酸，DTPA）标记，以聚l -赖氨酸为大分子载体。标记程序的主要优点是在不修改单链DNA杂交序列的情况下实现高度标记。通过DTPA的螯合基团与铕离子过滤杂交后，标记特定的杂交体。用时间分辨荧光法定量检测铕离子。检测靶DNA的灵敏度在低皮图范围内，与放射性标记的DNA探针相当。
A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.