SEQUENCE-SPECIFIC CHEMICAL NUCLEASES FOR GENOME ANALYSIS

为了促进基因组图谱和测序，有三种方法 靶向1，10-核酸酶的化学活性 邻菲咯啉-铜(OP-铜)对任何选定的DNA序列将被开发。 在第一种策略中，RNA从目标DNA序列转录而来 用5-烯丙胺-UTP代替UTP，然后用化学修饰 1，10-二氮杂菲。衍生的RNA用于形成R环，该R环 为裂解反应提供序列特异性，这是 通过添加铜离子、硫醇和过氧化氢来活化。在……里面 第二种方法，靶向切割的序列的一条DNA链 在基因组内是通过引物延伸替代合成的 5-烯丙基胺dUTP取代TTP。单链DNA被衍生化 以1，10-二氮杂菲为底物促进的RECA 链侵入靶序列，形成D-环。乳沟 然后，如上所述地激活反应。最后，cro蛋白将是 通过定点突变修饰以靶向核酸酶活性 OP-Cu.这三个程序都可以用来开发特定于序列的 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.