摘要
目的为探讨Taq DNA聚合酶(后简称Taq酶)中5′~3′外切酶活性在Taqman探针法荧光定量PCR中的作用及影响,指导热启动Taq酶性能改造,本文研究了不同热启动Taq聚合酶的5′~3′DNA外切酶活性差异,以及该活性的高低对Taqman探针法荧光定量PCR的影响。方法使用荧光探针为底物,分别测定不同热启动Taq酶的聚合酶活性及外切酶活性,对比在相同聚合酶活性下外切酶活性的差异,以及在荧光定量PCR扩增反应中的差异。结果在聚合酶活性相等的条件下,不同的热启动Taq聚合酶具有不同的5′~3′DNA外切酶活性,经化学修饰的Taq酶的5′~3′DNA外切酶活性分别下降2.7倍和4.55倍,经抗体修饰的Taq酶5′~3′DNA外切酶活性没有变化。在乙型肝炎病毒(HBV)DNA荧光定量PCR检测体系中,加入同等聚合酶活性单位的不同修饰方法制备热启动Taq酶,外切酶活性高的酶比活性低的酶ct值更低。结论在Taqman探针法荧光定量PCR反应中,Taq酶对探针的外切降解过程是反应的限速步骤,提高Taq酶5′~3′DNA外切酶活性有利于提高反应效率。
Objective In order to explore the role and influence of 5′~3′exonuclease activity in Taq polymerase in Taqman probe fluorescence quantitative PCR,and to guide the performance of hot⁃start Taq enzymes.To investigate the differences in 5′~3′DNA exonuclease activity for different hot⁃start Taq polymer⁃ases and the effect of the activity on the Taqman probe quantitative PCR.Methods Fluorescent probes were used as substrates to determine the polymerase activity and exonuclease activity of different hot⁃start Taq en⁃zymes,and to compare the difference in exonuclease activity and Q⁃PCR amplification efficiency under the same polymerase activity condition.Results Under the same conditions of polymerase activity,different hot⁃start Taq polymerases have different 5′~3′exonuclease activities.The 5′~3′DNA exonuclease activity of the chemically modified Taq enzyme decreased by 2.7 times and 4.55 times,respectively.The antibody⁃modified Taq enzyme 5′~3′DNA exonase activity did not change.In the Hepatitis B virus(HBV)DNA fluorescent quantitative PCR detection system,different modification methods with equivalent polymerase active units were added to prepare hot⁃start Taq enzymes.Enzymes with high exonuclease activity have lower ct values than enzymes with lower activity.Conclusion The exo⁃degradation of the probe is the rate⁃limiting step of the Taqman Q⁃PCR reaction,and increasing the 5′~3′exonuclease activity of the Taq enzyme can improve the reaction efficiency.
作者
蒋析文
刘霭珊
陈巍
JIANG Xiwen;LIU Aishan;CHEN Wei(Research Center of Medical and Pharmaceutical Bioengineering,Minstry of Health,National and Regional Joint Engineering Laboratory for Clinical Medical Molecular Diagnostics,Guangdong Province Nucleic Acid Molecular Diagnostics Engineering Technology Research Center,Guangdong Provincial Clinical Medical Mo⁃lecular Diagnostics Engineering Technology Center,DAAN Gene Co.,Ltd.Of Sun Yat⁃sen University,Guangzhou,Guangdong,China,510665)
出处
《分子诊断与治疗杂志》
2020年第2期170-174,243,共6页
Journal of Molecular Diagnostics and Therapy
基金
国家科技重大专项(2018ZX10732401,2018ZX10306414)。
