单引物耐热链替代扩增HBV X区方法的初步探讨

Amplification of HBV X region by thermophilic strand displacement amplification (SDA) with a typical SDA primer

目的 :以HBVX区为模型 ,对单引物耐热链替代扩增 (SDA)反应的影响因素进行初步探讨。SDA是一种等温的体外核酸扩增技术 ,其主要依据限制性内切酶识别并切割半硫酸磷酸化DNA未修饰链 (即打缺口 )以及 5′→ 3′外切酶缺陷的DNA聚合酶在切口处聚合延伸并替代下游链的特性 ,这种“打缺口—聚合 替代”不断循环往复 ,达到靶序列的扩增。方法 :以HBV为模型 ,设计一条典型的SDA引物 (含 5′悬端、BsoBⅠ酶切位点及 3′端靶序列互补区 ) ,采用耐热BsoBⅠ BstDNA聚合酶 5 3℃恒温反应体系 ,微孔板杂交检测最终产物。结果与结论 :对SDA反应的重要的影响因素进行了初步探讨 。

Objective:To discuss the main factors influencing thermophilic strand displacement amplification (SDA) with a typical primer. SDA is a technique of isothermal in vitro amplification of DNA, based on the ability of restriction enzyme to nick the unmodified strand of a hemiphosphorothioate DNA recognition sites, and the ability of a 5′→3′ exonuclease_deficient DNA polymerase to extend the 3′ end at the nick and displace the downstream strand. Target amplification is achieved by nicking and polymerization/displacement cycles.Methods:A typical SDA primer was designed on HBV X region DNA sequence. A reaction was conducted with thermophilic Bso BⅠ/ Bst DNA polymerase isothermally and its products were detected by microplate_based hybridization method.Results and Conclusion:The linear amplification of X region of HBV with the thermophilic SDA was achieved.The important factors influencing thermolphilic SDA were demonstrated.