SEQUENCE-SPECIFIC CLEAVAGE OF SINGLE-STRANDED-DNA - OLIGODEOXYNUCLEOTIDE-EDTA.FE(II)
SEQUENCE-SPECIFIC CLEAVAGE OF SINGLE-STRANDED-DNA - OLIGODEOXYNUCLEOTIDE-EDTA.FE(II)
复制标题
DOI：
10.1073/pnas.82.4.968
复制
发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
DERVAN, PB
中科院分区：
文献类型：
--
作者：
DREYER, GB;DERVAN, PB
本文描述了一种长度为19个核苷酸的DNA杂交探针的合成，该探针在第10位胸苷的C - 5位配备了金属螯合剂乙二胺四乙酸（用T*表示）。DNA - EDTA 1的序列为5′ - T - A - A - C - G - C - A - G - T* - C - A - G - G - C - A - C - C - G - T - 3′，它与质粒pBR322中的一个19核苷酸序列互补。在亚铁离子（Fe(II)）、氧气和二硫苏糖醇存在的情况下，DNA - EDTA 1在热变性的167碱基对限制性片段中其互补序列处发生特异性切割（25℃，pH 7.4，60分钟）。切割发生在探针1杂交位点的16个核苷酸范围内，可能是由于一种可扩散的活性物质。在167碱基对限制性片段中未观察到其他切割位点。用于合成DNA - EDTA探针的方法是基于掺入在C - 5位被乙二胺四乙酸三乙酯修饰的胸苷。通过使用常规的亚磷酰胺方法，胸苷 - EDTA可以被掺入到任何所需长度和序列的寡脱氧核苷酸中。由于DNA切割反应的效率取决于亚铁离子和还原剂（二硫苏糖醇）的添加，因此切割反应的起始可以被控制。这些DNA - EDTA·Fe(II)探针在温和条件下对单链DNA（很可能还有RNA）的序列特异性切割应该是有用的。
The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5''-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3'', which is complemetary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2 and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25.degree. C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA .cntdot. Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions.