一种DNA扩增的新技术:利用热稳定的Bst DNA聚合酶驱动跨越式滚环等温扩增反应  

A New Method: Themostable Bst DNA Polymerase Drives Saltatory Rolling Circle Amplification

作　　者：孟兆祥[1,2] 张伟[1] 檀建新[1] 马晓燕[1] 

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]中国检验检疫科学研究院食品风险管理与应用研究所,北京100123

出　　处：《中国生物化学与分子生物学报》2013年第9期892-898,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31371772);河北省自然科学基金项目(No.C2008000216)资助~~

摘　　要：Bst DNA聚合酶具有热稳定性、链置换活性及聚合酶活性,在体外DNA等温扩增反应中起重要作用.本文利用Bst DNA聚合酶的5'→3'聚合酶、核苷酸(末端)转移酶及链置换酶活性发展了一种新的体外环式DNA扩增技术跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA).在SRCA反应中,Bst DNA聚合酶以上游引物P1为模板合成其互补链RcP1,并和P1形成双链DNA;之后,Bst DNA聚合酶用其核苷酸转移酶活性在其P1的3'末端沿5'→3'方向随机掺入脱氧核糖核苷酸聚合形成寡聚核苷酸(dNMP)m序列,即DNA的合成反应跨越了RcP1与下游引物P2之间的缺口;然后,以下游引物P2为模板形成互补序列(RcP2);接着,Bst DNA聚合酶继续将脱氧核糖核苷酸随机添加到RcP2的3'末端,形成(dNMP)n序列.继而,Bst DNA聚合酶以RcP1为模板,继续催化聚合反应合成互补新链,并通过其链置换酶活性替换P1;如此往复,形成[P1-(dNMP)m-RcP2-(dNMP)n-…]序列.本文通过电泳、酶切、测序等方法对扩增产物进行分析,演绎出上述扩增过程,并就工作原理进行了讨论.该反应可能对开发等温扩增技术检测微生物有一定助益,也为解释环介导等温扩增技术中假阳性反应和滚环等温扩增反应中的背景信号提供了线索.Bst DNA polymerase used in DNA in vitro isothermal amplification has activities of both strand replacement and thermal stable DNA polymerization. In vitro roiling circle amplification technology has been developed with the combination of the 5＇→3＇ DNA polymerase activity, the strand displacement amplification activity and the terminal deoxynucleotidyl transferase （TdT） activity of Bst. We experimented on a sahatory rolling circle amplification （SRCA） model, where first, a complementary DNA strand for primer P1 （ RcP1 ） was synthesized by the polymerase activity and formed a double strand DNA （P1/RcP1）;then an oligonucleotide （dNMP）m of several deoxyribonucleotides were added to the 3＇ end of P1 by TdT activity ; as the complementary strand （RcP2） of primer （P2） was synthesized, BstDNA polymerase could ＂jump＂ the gap between P1 and RcP2; next, another oligonucleotide （dNMP）n was synthesized in a same way, where （dNMP）m could be added to allow continuous amplification involving the strand displacement activity; and lastly, the product of [ P1-（dNMP） m- RcP2-（dNMP） n- …] could be generated. In agarose gel electrophoresis of enzymatic digestion and DNA sequencing, we verified the SRCA products from its principle. Such assays might facilitate the detection of microorganisms by isothermal amplification. It might also help to explain the origin of background signal occurs in rolling circle amplification （RCA） and the false products in the reaction of loop-mediated isothermal amplification （LAMP）.

关 键 词：BST DNA聚合酶 跨越式滚环等温扩增 DNA扩增 

分 类 号：Q78[生物学—分子生物学] Q81