原核与真核生物宿主DNA残留检测方法的建立与比较

Comparative Study on the Method of Residual DNA in Prokaryotic and Eukaryotic Host

目的建立原核生物[大肠杆菌(E.coli)]和真核生物(酵母菌和CHO细胞)宿主DNA残留的检测方法,并比较检测方法的灵敏度。方法以E.coli、酵母菌和CHO细胞宿主DNA为模板,制备地高辛标记的探针。将探针与宿主DNA进行杂交后采用底物显色法显色,建立3种生物宿主DNA残留检测方法,并比较温度对检测方法灵敏度的影响。结果建立了E.coli、酵母菌和CHO细胞宿主DNA的检测方法,灵敏度高达10 pg;同一条件下真核生物宿主DNA残留检测方法的灵敏度要高于原核生物。结论该检测方法具有灵敏度高、准确性好、操作简便等优势,可用于E.coli、酵母菌和CHO细胞宿主DNA残留的检测及质量控制。

OBJECTIVE To establish the method for determination of residual genomic DNA in prokaryotic（ Escherichia coli）and eukaryotic（ yeast and CHO cells） host and compare the sensitivity of the test method. METHODS The total DNA of E.coli,yeast and CHO cells were labled with digoxin and used as a probe for substrate staining with host DNA. A method for the determination of residues genomic DNA in three organisms was established. And then the influence of different temperature on the sensitivity of the test method was compared. RESULTS The method for determination of residual genomic DNA in E. coli,yeast and CHO cells was established,and the minimum detection limit was 10 pg DNA.Under the same condition,the DNA method for determination of eukaryotes residual host DNA was much more sensitive than prokaryotes. CONCLUSION The method of solid phase dot blot hybridization is sensitive,accurate and easy operation,which is a good guiding significance in the production and quality control of recombination protein extracted from E.coli,yeast and CHO cells.