基础医学与临床 ›› 2016, Vol. 36 ›› Issue (7): 879-885.

• 研究论文 •    下一篇

自组装短肽K2I4K2在PCR中提高Taq DNA 聚合酶热稳定性

李萌萌,徐晓帆,陈振银,张慧楠,罗忠礼   

  1. 重庆医科大学
  • 收稿日期:2015-09-28 修回日期:2015-12-03 出版日期:2016-07-05 发布日期:2016-06-22
  • 通讯作者: 罗忠礼 E-mail:Zhongliluo@163.com
  • 基金资助:
    国家自然科学基金;重庆市自然科学基金项目;高等学校博士学科点专项科研基金项目

Self-assembling peptide K2I4K2 enhances the thermal-stability of Taq DNA polymerase in PCR system

  • Received:2015-09-28 Revised:2015-12-03 Online:2016-07-05 Published:2016-06-22

摘要: 目的 设计自组装短肽K2I4K2作为新型表面活性剂来稳定Taq DNA聚合酶,探索其对Taq DNA聚合酶在高温下的半衰期以及对PCR扩增效率的影响,探讨其在PCR中应用的可行性。方法 圆二色仪检测短肽K2I4K2在不同理化条件下的二级结构;原子力显微镜检测其自组装形成的纳米结构特征;紫外可见光谱检测其对Taq酶在高温下半衰期的影响;普通PCR验证其在PCR中应用的可行性;RT-qPCR检测其对Taq酶扩增效率的影响。结果 短肽K2I4K2在盐离子溶液中能自组装形成纳米纤维结构,且在高温下拥有稳定的二级结构;短肽K2I4K2 使得Taq在95℃下的半衰期增加了约1.6倍;短肽K2I4K2组装24 h后大大增加了Taq酶的扩增效率。结论 短肽K2I4K2 较为显著地增加了Taq DNA聚合酶的热稳定性,能够很好地应用于PCR反应系统中。

关键词: 自组装短肽, 纳米纤, Taq酶半衰期, PCR扩增效率

Abstract: Objective: To design a self-assembling peptide K2I4K2 as a new type of surfactant to stabilize Taq DNA polymerase, to explore the half-life of Taq DNA polymerase at high temperature and the influence on PCR amplification efficiency. Methods: Circular dichroism spectrum was used to detect the secondary structure of short peptide K2I4K2 in different physical and chemical condition. Atomic force microscope was used to detect the nanostructure characteristics formed by K2I4K2. UV-vis spectrum was used to examine the impact on the half-life of Taq enzyme stabilized by K2I4K2 at high temperature. Ordinary PCR was used to verify the feasibility of the application of K2I4K2 in the PCR. RT-qPCR was used to detecte of K2I4K2’s effect on the amplification efficiency of Taq polymerase. Results: Peptide K2I4K2 can form nanofiber structure in salt solution, and owns stable secondary structure at high temperature. UV-vis spectrum result indicated that peptide K2I4K2 increases the half-life by 1.6-fold compared with the stability in buffer alone. Furthermore, RT-qPCR indicated that peptide K2I4K2 greatly increase the amplification efficiency of Taq polymerase. Conclusion: Peptide K2I4K2 more significantly increased the thermal stability of the Taq DNA polymerase at a high temperature, and can be well used in the PCR system.

Key words: self-assembling peptide, nanofiber, half-life of Taq DNA polymerase, amplification of PCR

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