RecA蛋白介导的三链核酸结构形成及其序列提取(英文)

RecA-mediated Triple-stranded DNA Structure Formed by 100bp dsDNA and Extracted by Affinity Capture

人工合成的单链DNA分子经PCR扩增形成双链DNA分子。将RecA蛋白与生物素标记的寡聚核酸探针序列在ATPγS存在的情况下共同哺育,使RecA蛋白包裹寡聚核酸探针,然后加入含同源序列的上述双链DNA分子经适当环境哺育形成了稳定的局部三链核酸结构。通过加入链亲和素包裹的磁珠吸附生物素化的探针,这样同源双链DNA分子与寡聚核酸探针形成的局部三链核酸结构也被吸附在磁珠上。使用磁分离装置提取这一结构,逐步降低盐离子浓度以洗脱双链DNA分子。将洗脱液中残留的蛋白质去除,经PCR扩增可获得目的DNA序列。同时使用同源探针和非同源探针在其它序列中提取目的DNA序列,结果显示目的DNA序列只被同源探针提取。实验结果显示了这一三链核酸结构形成的序列特异性,并且其稳定性随盐离子浓度降低而下降。提示在这一结构中同源的寡聚核酸单链与双链DNA分子形成了氢键结合,同时提示使用文中描述的方法可以提取特异的序列,用以克隆相应的基因。

A sequence of single-stranded DNA molecule was synthesized and PCR amplified. Then sequence-specific recognition of double-stranded DNA by its homologous deoxyoligonueleotides, which were coated with RecA protein, was performed in the presence of the non-hydrolyzable analog of ATP--ATPγS. After the reaction, local stable triple-stranded DNA structure was formed. By using affinity capture, the target double- stranded DNA strands were retained together with the biotinylated deoxyoligonucleotide probes and extracted. In the same way, the target DNA sequence can only be extracted by using homologous deoxyoligonueleotides probe. It demonstrates that the recognition is sequence specific. The observations also show that the RecA-mediated triple-stranded DNA structure can be dissociated with the gradient decrease of saline concentration. It is suggested that hydrogen bonds are formed between incoming strand and intrinsic double-stranded DNA and the methods can be used to isolate special DNA sequences conveniently and efficiently.