SEQUENCE-SPECIFIC CHEMICAL NUCLEASES FOR GENOME ANALYSIS

为了促进基因组作图和测序， 用于靶向1，10- 将开发与任何选定的DNA序列相匹配的菲咯啉-铜（OP-Cu）。 在第一种策略中，RNA从靶DNA序列转录 使用5-烯丙基胺-UTP代替UTP，然后用 1，10-菲咯啉 衍生的RNA用于形成R环， 为断裂反应提供了序列特异性， 通过加入铜离子、硫醇和过氧化氢来活化。 在 第二种方法，切割靶序列的一条DNA链， 在基因组内通过引物延伸取代合成 5-烯丙胺dUTP代替TTP。单链DNA被衍生化， 与1，10-菲咯啉，并用作Rec A的底物， 链侵入靶序列以形成D环。 裂解 然后如上所述激活反应。 最后，cro蛋白将被 通过定点诱变修饰以靶向 OP-Cu。 所有这三种方法都可以用来开发序列特异性的 任何预选序列的核酸酶。

In order to facilitate genome mapping and sequencing, three approaches for targeting the chemical nuclease activity of 1,10- phenanthroline-copper(OP-Cu) to any chosen DNA sequence will be developed. In the first strategy, RNA is transcribed from the target DNA sequence using 5-allylamine-UTP in place of UTP and then chemically modified with 1,10-phenanthroline. The derivatized RNA is used to form an R-loop that provides the sequence specificity for the scission reaction which is activated by the addition of cupric ion, thiol and hydrogen peroxide. In the second method, one DNA strand of the sequence targeted for cleavage within the genome is synthesized by primer extension substituting 5-allylamine dUTP in place of TTP. .The single-strand DNA is derivatized with 1,10-phenanthroline and used as a substrate for Rec A facilitated strand invasion of the target sequence to form a D-loop. The cleavage reaction is then activated as above. Finally, the cro protein will be modified by site-directed mutagenesis to target the nuclease activity of OP-Cu. All three procedures could be used to develop sequence-specific nucleases for any preselected sequence.