滚环扩增阳离子共轭聚合物均相检测microRNA

doi:10.3969/j.issn.1000-1565.2013.03.007

河北大学学报(自然科学版) ›› 2013, Vol. 33 ›› Issue (3): 258-263.DOI: 10.3969/j.issn.1000-1565.2013.03.007

滚环扩增阳离子共轭聚合物均相检测microRNA

成永强,贾海莲,唐志远,赵晶晶,李正平

河北大学化学与环境科学学院,药物化学与分子诊断省部共建教育部重点实验室,河北保定071002

出版日期:2013-05-25 发布日期:2013-05-25
基金资助:
国家自然科学基金资助项目，高等学校博士点基金资助项目，河北省自然科学基金资助项目

Homogeneous detection of microRNA by rolling circle amplification and cationic conjugated polymer

CHENG Yongqiang,JIA Hailian,TANG Zhiyuan,ZHAO Jingjing,LI Zhengping

Online:2013-05-25 Published:2013-05-25

摘要/Abstract

摘要： 结合恒温滚环扩增(rolling circle amlification,RCA)、单链特异性核酸外切酶工(exonuclease Ⅰ,Exo Ⅰ)和阳离子共轭聚合物(Cationic conjugated polymer,CCP)荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术,建立了一种特异、灵敏的均相检测microRNA(miRNA)的新方法.该方法应用荧光标记探针与RCA扩增的长链DNA产物杂交,当加入CCP时,其与杂交的标记探针通过静电力结合,发生高效的FRET.未杂交的标记探针利用Exo Ⅰ水解成单核苷酸,其与CCP相互作用力弱,不能发生有效的FRET.基于此,无需分离和洗涤步骤,实现了RCA扩增miRNA的均相检测.方法特异性好,灵敏度高,线性为0.5～20 pmol/L,检出限为0.2 pmol/L.方法为miRNA均相检测和原位成像分析以及临床诊断提供了新策略.

关键词: 分析化学, microRNA, 滚环扩增, 阳离子共轭聚合物, 荧光共振能量转移(FRET)

中图分类号:

O657.34

成永强,贾海莲,唐志远,赵晶晶,李正平. 滚环扩增阳离子共轭聚合物均相检测microRNA[J]. 河北大学学报(自然科学版), 2013, 33(3): 258-263.

CHENG Yongqiang,JIA Hailian,TANG Zhiyuan,ZHAO Jingjing,LI Zhengping. Homogeneous detection of microRNA by rolling circle amplification and cationic conjugated polymer[J]. Journal of Hebei University (Natural Science Edition), 2013, 33(3): 258-263.

参考文献

[1] HE Lin, HANNON G J. MicroRNAs:small RNAs with a big role in gene regulation [J]. Nature Reviews Genetics 2004, 5.
[2] Hammond SM. microRNA detection comes of age [J]. Nature methods 2006, 1(1).
[3] Mitchell, PS, Parkin, RK, Kroh, EM, Fritz, BR, Wyman, SK, Pogosova-Agadjanyan, EL, Peterson, A, Noteboom, J, O'Briant, KC, Allen, A. Circulating microRNAs as stable blood-based markers for cancer detection [J]. Proceedings of the National Academy of Sciences of the United States of America 2008, 30(30).
[4] le Sage C, Nagel R, Egan DA, Schrier M, Mesman E, Mangiola A, Anile C, Maira G, Mercatelli N, Ciafre SA. Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation [J]. EMBO Journal 2007, 15(15).
[5] Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR. Real-time quantification of microRNAs by stem-loop RT-PCR [J]. Nucleic Acids Research 2005, 20(20).
[6] Lizardi PM., Zhu ZR., Bray-Ward P., Thomas DC., Ward DC., Huang XH.. Mutation detection and single-molecule counting using isothermal rolling-circle amplification [J]. Nature Genetics 1998, 3(3).
[7] Yongqiang Cheng, Xian Zhang, Zhengping Li. Highly Sensitive Determination of microRNA Using Target-Primed and Branched Rolling-Circle Amplification [J]. Angewandte Chemie 2009, 18(18).
[8] Yao B, Li J, Huang H. Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification. [J]. RNA 2009, 9(9).
[9] XINRUI DUAN, LIBING LIU, FUDE FENG. Cationic Conjugated Polymers for Optical Detection of DNA Methylation, Lesions, and Single Nucleotide Polymorphisms [J]. Accounts of Chemical Research 2010, 2(2).
[10] Zhang, J., Li, Z., Wang, H., Wang, Y., Jia, H., Yan, J.. Ultrasensitive quantification of mature microRNAs by real-time PCR based on ligation of a ribonucleotide-modified DNA probe [J]. Chemical communications 2011, 33(33).
[11] FENG Fude, HE Fang, AN Lingling. Fluorescent conjugated polyelectrolytes for biomacromolecule detections [J]. Advanced Materials 2008, 20.

滚环扩增阳离子共轭聚合物均相检测microRNA

Homogeneous detection of microRNA by rolling circle amplification and cationic conjugated polymer

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价