非变性聚丙烯酰氨凝胶电泳及银染法临床应用探讨

Clinical application of non denaturant polyacrylamide gel electrophoresis and silver staining test

目的:建立一种省时简便和灵敏可靠的检测筛查基因点突变的方法,即PCR-SSCP(PCR-Singlestrandconformationpolymorphism)银染分析技术。方法:应用改进的PCR-SSCP银染分析技术,检测妊娠期胆内胆汁淤积症患者外周血基因组DNA中MDR3基因外显子14的点突变情况。结果:非变性聚丙烯酰氨凝胶电泳在有冷水循环装置控制下,结果稳定可靠,受环境温度影响小;但银染时,需根据室温调整染色时间及显影液的配制,当室温高于25℃时,碳酸钠需预冷,有利于控制背景色,得到更为满意的结果。结论:利用非变性聚丙烯酰氨凝胶电泳及银染法筛查目的基因的点突情况,具有省时简便和灵敏可靠的特点,且重复性好,避免了盲目测序及资金浪费。

Objective To explore a save time and reliable newly method for detecting and screening gene point mutation, namely PCR Single strand conformation polymorphism (PCR SSCP) silver staining analysis technique. Methods PCR SSCP technique improved was applied to detect exon 14 of the MDR3 gene point mutation condition in peripheral blood of patients with intrahepatic cholestasis of pregnancy(ICP) from Jan. 2004 to June 2004. Results With cold water cycle equipment under controlling of non denaturant polyacrylamide gel electrophoresis, the results remained stable and credible and the ambient temperature had been less influenced; But when silver staining test was used, concentration of developer and the room temperature had to be taken into consideration. When the room temperature reached a higher point ( >25℃),sodium carbonate required precooling, which could be helpful to control its background color with a satisfactory outcome. Conclusion The test can be save time and be reliable for detecting and screening gene mutation condition, moreover it can be well duplication at the same condition and avoid blindness sequencing and fund wasting.