凝胶迁移阻滞法分析人类端粒酶的DNA结合特性

Testing the property for DNA-binding of human telomerase by the gel mobility shift assay

目的采用凝胶迁移阻滞法分析研究人类端粒酶DNA结合特性。方法以地高辛标记的人端粒序列寡核苷酸与人端粒酶提取物进行结合反应,以未参与结合反应的单纯标记寡核苷酸作为参照。实验所设的3组样品中,第一组和第二组标记寡核苷酸加端粒酶,第一组显示一条阻滞性条带,第二组显示两条条带,前方的一条与第一组条带平行,为单纯探针条带,后方的一条迁移率被阻滞,应为探针与纯化的蛋白复合体相结合的结果;第三组为标记寡核苷酸加端粒酶阴性洗涤液,只显示单一探针条带。结果只有人端粒序列寡核苷酸与人端粒酶提取物结合反应管显示特异的凝胶迁移阻滞性电泳条带。结论人端粒酶具有和端粒DNA片段结合的特性。

Objective The specific property for DNA-binding of human telomerase was tested by gel mobility shift assay.Methods Human telomerase (hTR) sequence fragments oligonucleotide marked by digoxin in binding reaction with telomerase extract was compared with that without binding reaction. Of three samples groups, the labeled hTR and oligonucleotide in groupⅠandⅡ, a mobility shift band appeared in groupⅠ; two bands appeared in groupⅡ, one as simplex probe band paralleling with that of groupⅠ, the other one with blocked morbility as the result of combination between probe and purified protein complex; then the negative scrub solution of labeled hTR oligonucleotide in group Ⅲ, only a probe band appeared.Results Only the binding reaction tube with hTR oligonucleotide and hTR extracts could show specific gel mobility shift electrophoresis band. Conclusions hTR should have specific property for DNA-binding.