|本期目录/Table of Contents|

[1]邓世琼,董娟,陈刚毅,等.基于缺口酶的链置换等温扩增技术[J].应用与环境生物学报,2015,21(06):1080-1085.[doi:10.3724/SP.J.1145.2015.03039]
 DENG Shiqiong,DONG Juan,CHEN Gangyi,et al.Nicking enzyme-based isothermal strand displacement amplification[J].Chinese Journal of Applied & Environmental Biology,2015,21(06):1080-1085.[doi:10.3724/SP.J.1145.2015.03039]
点击复制

基于缺口酶的链置换等温扩增技术()
分享到:

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
21卷
期数:
2015年06期
页码:
1080-1085
栏目:
研究论文
出版日期:
2015-12-25

文章信息/Info

Title:
Nicking enzyme-based isothermal strand displacement amplification
作者:
邓世琼 董娟 陈刚毅 杜凤 邹家尉 唐卓
中国科学院成都生物研究所 成都 610041
Author(s):
DENG Shiqiong DONG Juan CHEN Gangyi DU Feng ZOU Jiawei TANG Zhuo
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
关键词:
缺口酶等温扩增剪切链置换扩增
Keywords:
nicking enzyme isothermal amplification nick strand displacement amplification
分类号:
Q5-3
DOI:
10.3724/SP.J.1145.2015.03039
文献标志码:
A
摘要:
传统的链置换等温扩增技术中需要加入修饰的底物,为了解决这一技术难题,实现常规底物下的链置换等温扩增技术,采用缺口酶Nt.BstNBI代替传统链置换等温扩增技术中的限制性内切酶,通过优化两种酶的比例、反应温度、反应时间、两种原装buffer的比例、buffer中的离子浓度等反应条件和引物浓度、引物序列以及引物前端保护序列的长度,最终建立了一种新型的链置换等温扩增技术. 该方法可以检测到2 pmol/L的质粒DNA. 扩增反应在15-30 min即可完成,并且产物可通过琼脂糖凝胶电泳进行直接检测. 本研究建立的基于缺口酶的链置换等温扩增技术较之传统方法更加简便、快捷、环保且价格低廉,具有非常强的实际应用潜力,特别适用于现场检测.
Abstract:
Modified substrate (dATPαS) is needed in traditional isothermal strand displacement amplification. In order to realize isothermal strand displacement amplification at conventional substrate, this study replaced the restriction enzyme in traditional isothermal strand displacement amplification with nicking enzyme Nt.BstNBI. The detection limit of plasmid DNA was as low as 2 pmol/L. The amplification reaction could be completed in 15-30 min, with products directly detectable by agarose gel electrophoresis. The results showed that the nicking enzyme-based isothermal strand displacement amplification is an environment-friendly, cost effective new technology that is faster, more convenient, and practical than the traditional method, especially for on-site testing.

参考文献/References:

1 Compton J. Nucleic acid sequence-based amplification [J]. Nature, 1991, 350: 91-92
2 Fire A, Xu SQ. Rolling replication of short DNA circles [J]. Proc Natl Acad Sci, 1995, 92: 4641-4645
3 Vincent M, Xu Y, Kong HM. Helicase dependent isothermal DNA amplification [J]. Sci Rep, 2004, 5: 795-800
4 Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products [J]. Nat Protocols, 2008, 3: 877-882
5 Gill P, Ghaemi A. Nucleic acid isothermal amplification technologies - A review [J]. Nucleosides Nucleotides Nucleic Acids, 2008, 27: 224-243
6 Gao WJ, Zeng LW, Peng T. Rapid isothermal detection assay: a probe amplification method for the detection of nucleic acids [J]. Diagnostic Microbiol Infectious Dis, 2008, 60: 133-141
7 Walker GT, Fraiser MS, Schram JL, Little MC, Nadeau JG, Malinowski DP. Strand displacement amplification-an isothermal, in vitro DNA amplification technique [J]. Nucleic Acids Res, 1992, 20: 1691-1696
8 Li J, Young CS, Lizardi PM, Stern DF. In situ detection of specific DNA double strand breaks using rolling circle amplification [J]. Cell Cycle, 2005, 4: 1767-1773
9 Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers [J]. Mol Cell Probe, 2002, 16: 223-229
10 赵永云, 杜凤, 董娟, 陈浩东, 周丽, 陈蓉, 唐卓. 孔雀石绿和结晶紫在荧光检测中的比较[J]. 应用与环境生物学报, 2013, 19 (2) : 365-369 [Zhao YY, Du F, Dong J, Chen HD, Zhou L, Chen R, Tang Z. Comparison of two triphenylmethane dye (malachite green and crystal violet) in fluorescence detection [J]. Chin J Appl Environ Biol, 2013, 19 (2): 365-369]

备注/Memo

备注/Memo:
国家自然科学基金项目(21402189,21322208)和四川省科技支撑计划项目(苗子工程,2014RZ0022)资助 Supported by the National Natural Science Foundation of China (21402189,?21322208),?and the Science and Technology Support Program of Sichuan Province (2014RZ0022)
更新日期/Last Update: 2016-01-05