物理化学学报 >> 2017, Vol. 33 >> Issue (10): 2052-2057.doi: 10.3866/PKU.WHXB201750105

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DNA的滚环扩增合成研究

刘淑贞,张志庆*(),王芳,周亭,王秀凤,张国栋,刘婷婷,张洪芝   

  • 收稿日期:2017-03-29 发布日期:2017-07-17
  • 通讯作者: 张志庆 E-mail:zhangzq@upc.edu.cn
  • 基金资助:
    教育部留学回国人员科研启动基金(2014010615);国家自然科学青年基金(21603276);国家自然科学青年基金(21303267);山东省自然科学基金(ZR2016BL14);中央高校基本科研业务费资助项目

Study on the Synthesis of DNA via Rolling Circle Amplification

Shu-Zhen LIU,Zhi-Qing ZHANG*(),Fang WANG,Ting ZHOU,Xiu-Feng WANG,Gou-Dong ZHANG,Ting-Ting LIU,Hong-Zhi ZHANG   

  • Received:2017-03-29 Published:2017-07-17
  • Contact: Zhi-Qing ZHANG E-mail:zhangzq@upc.edu.cn
  • Supported by:
    the Scientific Research Foundation for Returned Overseas Chinese Scholars(2014010615);National Natural Science Foundation of China(21603276);National Natural Science Foundation of China(21303267);Natural Science Foundation of Shandong Province, China(ZR2016BL14);Fundamental Research Funds for the Central Universities, China

摘要:

滚环扩增(RCA)反应作为一种简单高效的等温酶促反应,现已发展为核酸扩增领域的新技术,其产物在组装体搭建和多功能材料的制备方面有着广泛的应用。本文采用琼脂糖凝胶、紫外和透射电镜(TEM)等手段,探究了时间、三磷酸脱氧核糖核苷(dNTPs)、酶以及引物的浓度等因素对脱氧核糖核酸(DNA)滚环扩增产物的影响。结果表明:在反应开始的前30 min,RCA产物的长度受时间的影响比较明显;随着dNTPs浓度的提高,RCA产物的链长增长,浓度也不断提高;酶和引物的浓度对滚环扩增产物的长度没有明显影响,但对RCA产物浓度的影响较大,过量的酶致使RCA产物的含量显著下降。

关键词: 滚环扩增技术, 核酸, dNTPs, 聚合酶, 引物

Abstract:

Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic reaction, which has developed as a novel technology in the field of nucleic acid amplification. Its product has a wide range of applications in the assembly and preparation of multi-functional materials. Here, we report the effects of reaction time and the concentrations of deoxyribonucleoside triphosphates (dNTPs), polymerase, and primer on the product of RCA. The RCA product was characterized by methods including agarose gel electrophoresis, ultraviolet spectroscopy, and transmission electron microscopy (TEM). The results showed that the length of the RCA product was significantly affected by the reaction time, especially when the reaction time was less than 30 min. With an increase of dNTPs concentrations, the concentration and chain length of the RCA product increased. However, while the concentrations of enzyme and primer had little effect on the length of the RCA product, they had a large effect on its concentration. It is worth noting that the content of the RCA product decreased significantly in the presence of excess enzyme concentration.

Key words: Rolling circle amplification, Nucleic acid, dNTPs, Polymerase, Primer

MSC2000: 

  • O648