设计特定序列的引物提高依赖解旋酶恒温扩增效率  

Primers with Designed Terminal Sequence Improved Efficiency of Helicase-dependent DNA Amplification

作　　者：张培培 吴洋[1,2,3] 王玲 齐浩[1,2,3] ZHANG Pei-Pei;WU Yang;WANG Ling;QI Hao(Department of Pharmaceutical Engineering,School of Chemical Engineering,Tianjin University,Tianjin 300072;Key Laboratory of System Biotechnology of Ministry of Education,Tianjin 300072;Synthetic Biology Platform of Chemical Innovation Center of Tianjin University,Tianjin 300072)

机构地区：[1]天津大学化工学院制药工程系,天津300072 [2]系统生物工程教育部重点实验室,天津300072 [3]天津大学化工协同创新中心合成生物学平台,天津300072

出　　处：《中国生物化学与分子生物学报》2018年第7期747-753,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.21476167)~~

摘　　要：依赖解旋酶恒温扩增(helicase-dependent amplification,HDA)是一种新型的恒温DNA体外扩增方法,具有反应原理简单,反应温度恒定等优点,被广泛地应用于核酸分子检测。但是依赖解旋酶恒温扩增仍面临着双链DNA解旋反应效率不高导致的扩增效率不够高、灵敏度低的问题。为了进一步提高依赖解旋酶恒温扩增技术中双链DNA模板的解旋效率,在本研究中,5'端带有不同长度不同序列的寡聚核苷酸引物,被用于依赖解旋酶恒温扩增反应,以研究DNA片段的末端序列对依赖解旋酶的双链DNA解旋反应,乃至整个扩增反应效率的影响。结果表明,只有5'端带有poly(A)序列的核酸引物明显促进了扩增反应效率,其中6个A的长度的5'端组成的引物对恒温扩增的促进作用最好,扩增产物比不带任何特定序列的原引物提高了约5.3倍。相比之下,带有poly(AC)或者poly(C)序列的引物,在一定程度上阻碍依赖解旋酶恒温扩增反应效率。因此,本研究证明了特定设计的末端序列,可以提高依赖解旋酶恒温扩增反应效率。Helicase-dependent amplification（ HDA） is a new method of isothermal amplification. It has the advantages of simple reaction principle and constant reaction temperature and has been applied to amplify DNA fragment from DNA source including genomic DNA,plasmid DNA,and c DNA rapidly.However,due to the low activity of DNA helicase mediated amplification, there are still many weaknesses,especially insufficient amplification efficiency. To further enhance the efficiency of the helicase-dependent amplification,primers with different designed sequences at the 5’ terminal end were applied. The HDA products were quantified and compared with the amplification from primers without the designed sequence. The results demonstrated that the primer with poly（ A） sequence at 5’ terminal end improved the amplification effectively,the 6 × A tail resulted a 5. 3-fold increasing in amplification. In contrast,sequence of poly（ AC） or poly（ C） barely helped and instead decreased the amplification.Consequently,poly（ A） with optimized length can be designed at the terminus of DNA primers to improvethe efficiency of helicase-dependent amplification reaction.

关 键 词：依赖解旋酶恒温扩增 解旋酶 引物设计 

分 类 号：Q7[生物学—分子生物学]