植物学报 ›› 2018, Vol. 53 ›› Issue (1): 104-109.DOI: 10.11983/CBB16216

• 技术方法 • 上一篇    下一篇

利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段

曹媛, 杨云, 徐化全, 刘洋, 王丹阳*()   

  1. 西北大学生命科学学院, 西部资源生物与现代生物技术教育部重点实验室, 西安 710069
  • 收稿日期:2016-11-11 接受日期:2017-04-17 出版日期:2018-01-01 发布日期:2018-08-10
  • 通讯作者: 王丹阳
  • 基金资助:
    国家自然科学基金(No.31200234)和陕西省教育厅省级重点实验室科研计划(No.14JS089)

PCR Used to Find Plasmid Backbone Fragments in the Products of hiTAIL-PCR

Yuan Cao, Yun Yang, Huaquan Xu, Yang Liu, Danyang Wan*()   

  1. Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an 710069, China
  • Received:2016-11-11 Accepted:2017-04-17 Online:2018-01-01 Published:2018-08-10
  • Contact: Danyang Wan

摘要:

T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR (hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现, 利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列, 而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应, 用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中, 通过排除这些片段, 提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时, 通过调整反应程序, 使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana) T-DNA突变体drf1侧翼序列的克隆实例中, 对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条, 效率提高了81.8%。

关键词: hiTAIL-PCR, T-DNA突变体, 侧翼序列, 质粒骨架, 克隆

Abstract:

T-DNA mutants are important resources for research of gene function. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is widely used for cloning the flanking sequence near the T-DNA insertion sites. However, we found that some cloned flanking fragments in hiTAIL-PCR products corresponded not to the host genomic DNA but to the plasmid backbone DNA. In this study, with a control of the RB-S4/AC1 or LB-A4/AC1 product, we amplified PCR fragments from the plasmid backbone DNA. By excluding them from further analysis, we amplified fragments from the unknown genomic DNA more effectively. Meanwhile, by adjusting the PCR programs, the whole PCR time was greatly shortened. In cloning the flanking sequence of Arabidopsis thaliana T-DNA mutant drf1, our method with hiTAIL-PCR reduced the total 22 DNA bands required for further checking to 4 bands, which improved the efficiency by 81.8%.

Key words: hiTAIL-PCR, T-DNA mutant, flanking sequence, plasmid backbone, cloning