DNA足纹步移作图法

A Method of DNA Footprinting Walking Map

一种以PCR介导的、可对任意长度靶DNA片段上的核蛋白结合位点进行DNA足纹作图分析的新方法 .原理是 :采用被随机降解靶DNA分子作为模板 ,用标记的跨越整个模板的足够多条特异性引物进行单链扩增 .首先 ,利用某种化学试剂或酶如DNaseⅠ对已与蛋白质结合或未结合的双链靶DNA进行随机降解 ,在一定条件下使每一个DNA分子恰好只有一个位点被切割 .然后 ,这些被随机降解DNA分子即可作为模板 ,用同位素标记的跨越整个模板的足够多条特异性引物 (正向或反向 )进行单链扩增 .最后 ,扩增的单链产物通过变性聚丙烯酰胺凝胶电泳和放射自显影形成DNA片段梯队 ,而被蛋白质保护的位点则在DNA片段梯队中形成位置缺口 ,从而确定DNA与蛋白质相结合的精确位点 .该方法被应用对人干细胞因子基因 5′旁侧 - 1190～ - 2

A novel method was developed to prepare for PCR- mediated DNA footprinting walking map. This method is based on the amplification of the cleaved strands using multi-specific primers to walk along the whole DNA template. At first, long enough target DNA fragment without being labeled is incubated in the presence or absence of nuclear proteins and cleaved randomly on average once by either a chemical reagent or an enzyme such as DNase I. Then the labeled multi-specific primers (sense or antisense) are utilized to amplify the cleaved DNA template to produce labeled single strands which can walk along the whole template. At last, the single strand DNA is separated by electrophoresis in a denaturing polyacrylamide gel, followed by autoradiography analysis. The result is the appearance of a 'gap' in the sequence ladder of protein-combined DNA compared to the pattern of unbound or unprotected DNA. This method can footprint walking along any length of DNA template in one time theoretically if enough specific primers are utilized. This method was applied to investigate the partial footprinting map of 5 ' flanking region from -1190 to -273 of human stem cell factor gene.