RecA介导的特异性DNA片段捕获

外显子捕获联合高通量测序技术在检测新的致病基因,特别是罕见的基因变异时,表现出很高的检测效率.但在具体使用过程中,探针易出现非特异性杂交,设计探针时需考虑Tm值均一性、所需初始样品量较大等问题.RecA是原核生物同源重组的中心分子,参与DNA损伤的重组修复.通过在体外模拟RecA蛋白在原核生物体内重组寻找同源序列的途径,用以捕获目标DNA分子,以期提高外显子捕获过程中的探针杂交效率和特异性.根据RecA在体内同源重组中的作用模式,先将基因组染色质片段化,再纯化DNA,设计生物素标记的特异性探针,在RecA蛋白的介导下以捕获基因组中的目的同源片段.结果显示:设计的和目标片段互补的探针高效而特异地捕获了目标DNA片段,ATP和水能够破坏RecA介导形成的三链复合体的稳定性,可以作为很好的杂交后洗脱试剂,而且水直接作为洗脱试剂可以提高洗脱目的 DNA片段的效率和纯度.

RecA-Mediated Capture of Specific DNA

Exome sequencing technology combined with high-throughput sequencing provides a substantial improvement for detecting new disease-causing gene,especially for rare genetic mutation which shows high sensitivity.However,exon probes designing require the homogeneity of Tm,hybridization is prone to non-specific and the technology requires larger quantity of initial sample.Homologous recombination functioned in the repair of DNA break and crossover in the process of meiosis implies that it is a good strategy to capture DNA fragment in vitro.According to RecA mediated homologous recombination in vivo,we have broke genome,purified DNA and engaged biotin-labelled probes to capture specific homologous DNA fragment with the help of RecA.Our results show that probe complementary to targeted DNA can capture DNA specifically and efficiently.We also found that ATP and H2O can dissociate established triple-strand DNA complex.ATP and H2O will easily elute binding DNA with high efficiency and purity.