Please wait a minute...
物理化学学报  2017, Vol. 33 Issue (10): 2052-2057    DOI: 10.3866/PKU.WHXB201750105
论文     
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
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
 全文: PDF(1312 KB)   HTML 输出: BibTeX | EndNote (RIS) |
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
收稿日期: 2017-03-29 出版日期: 2017-05-10
中图分类号:  O648  
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
通讯作者: 张志庆     E-mail: zhangzq@upc.edu.cn
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
刘淑贞
张志庆
王芳
周亭
王秀凤
张国栋
刘婷婷
张洪芝

引用本文:

刘淑贞,张志庆,王芳,周亭,王秀凤,张国栋,刘婷婷,张洪芝. DNA的滚环扩增合成研究[J]. 物理化学学报, 2017, 33(10): 2052-2057, 10.3866/PKU.WHXB201750105

Shu-Zhen LIU,Zhi-Qing ZHANG,Fang WANG,Ting ZHOU,Xiu-Feng WANG,Gou-Dong ZHANG,Ting-Ting LIU,Hong-Zhi ZHANG. Study on the Synthesis of DNA via Rolling Circle Amplification. Acta Phys. -Chim. Sin., 2017, 33(10): 2052-2057, 10.3866/PKU.WHXB201750105.

链接本文:

http://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB201750105        http://www.whxb.pku.edu.cn/CN/Y2017/V33/I10/2052

Oligonucleotide Sequence (5??3?)
circular template DNA1 TGTCTTCGCCTTCTTGTTTCC
TTTCCTTGAAACTTCTTCCTT
TCTTTCTTTCGACTAAGCACC
primer DNA2 GGCGAAGACAGGTGCTTAGTC
表1  核苷酸序列
图1  时间对RCA产物的影响
图2  RCA产物的TEM图
图3  dNTPs摩尔浓度对RCA产物的影响
图4  phi29 DNA聚合酶对RCA产物的影响
图5  引物对RCA产物的影响
1 Ali M. M. ; Li F. ; Zhang Z. Q. ; Zhang K. X. ; Kang D. ; Ankrum J.A. ; Le X. C. ; Zhao W. A. Chem. Soc. Rev. 2014, 43, 3324.
doi: 10.1039/c3cs60439j
2 Terpe K. Appl. Microbiol. Biotechnol. 2013, 97 (24), 10243.
doi: 10.1007/s00253-013-5290-2
3 Konry T. ; Smolina I. ; Yarmush J. M. ; Irimia D. ; Yarmush M. L. Small 2011, 7 (3), 395.
doi: 10.1002/smll.201001620
4 Deng Z. X. ; Tian Y. ; Lee S. H. ; Ribbe A. E. ; Mao C. D. Angew. Chem. Int. Ed. 2005, 44, 3582.
doi: 10.1002/anie.200463096
5 Wilner O. I. ; Shimron S. ; Weizmann Y. ; Wang Z. G. ; Willner I. Nano Lett. 2009, 9 (5), 2040.
doi: 10.1021/nl900302z
6 Hamblin G. D. ; Carneiro K. M. M. ; Fakhoury J. F. ; Bujold K. E. ; Sleiman H. F. J. Am. Chem. Soc. 2012, 134 (6), 2888.
doi: 10.1021/ja2107492
7 Ouyang X. Y. ; Li J. ; Liu H. J. ; Zhao B. ; Yan J. ; Ma Y. Z. ; Xiao S. J. ; Song S. P. ; Huang Q. ; Jie Chao J. ; Fan C. H. Small 2013, 9 (18), 3082.
doi: 10.1002/smll.201300458
8 Yan J. ; Hu C. Y. ; Wang P. ; Liu R. ; Zuo X. L. ; Liu X. W. ; Song S. P. ; Fan C. H. ; He D. N. ; Sun G. ACS Appl. Mater. Interfaces 2014, 6 (22), 20372.
doi: 10.1021/am505913d
9 Linck L. ; Rei? E. ; Bier F. ; Resch-Genger U. Anal. Methods 2012, 4, 1215.
doi: 10.1039/c2ay05760c
10 Xue Q. W. ; Wang Z. G. ; Wang L. ; Jiang W. Bioconjug. Chem. 2012, 23 (4), 734.
doi: 10.1021/bc200537g
11 Akter F. ; Mie M. ; Grimm S. ; Nygren P?. ; Kobatake E. Anal. Chem. 2012, 84 (11), 5040.
doi: 10.1021/ac300708r
12 Zhu Y. ; Wang H. J. ; Wang L. ; Zhu J. ; Jiang W. ACS Appl. Mater. Interfaces 2016, 8 (4), 2573.
doi: 10.1021/acsami.5b10285
13 Zhao W. A. ; Cui C. H. ; Bose S. ; Guo D. ; Shen C. ; Wong W. P. ; Halvorsen K. ; Farokhzad O. C. ; Teo G. S. L. ; Philips J. ; Dorfman D. M. ; Karnik R. ; Karp J. M. Proc. Natl. Acad. Sci. (USA) 2012, 109, 19626.
doi: 10.1073/pnas.1211234109
14 Chen G. ; Liu D. ; He C. B. ; Gannett T. R. ; Lin W. B. ; Weizmann Y. J. Am. Chem. Soc. 2015, 137 (11), 3844.
doi: 10.1021/ja512665z
15 Zhang Z. Q. ; Ali M. M. ; Eckert M. A. ; Kang D. K. ; Chen Y. Y. ; Sender L. S. ; Fruman D. A. ; Zhao W. A. Biomaterials 2013, 34, 9728.
doi: 10.1016/j.biomaterials.2013.08.079
16 Zhao W. A. ; Gao Y. ; Kandadai S. A. ; Brook M. A. ; Li Y. F. Angew. Chem. Int. Ed. 2006, 45, 2409.
doi: 10.1002/anie.200600061
17 Wu Z. S. ; Zhou H. ; Zhang S. B. ; Shen G. L. ; Yu R. Q. Anal. Chem. 2010, 82 (6), 2282.
doi: 10.1021/ac902400n
18 Kim J. H. ; Jang M. ; Kim Y. J. ; Ahn H. J. J. Med. Chem. 2015, 58 (19), 7863.
doi: 10.1021/acs.jmedchem.5b01126
19 Ye T. ; Chen J. Y. ; Liu Y. F. ; Ji X. H. ; Zhou G. H. ; He Z. K. ACS Appl. Mater. Interfaces 2014, 6 (18), 16091.
doi: 10.1021/am504035a
20 Chen A. Y. ; Ma S. Y. ; Zhou Y. ; Chai Y. Q. ; Yuan R. Anal. Chem. 2016, 88 (6), 3203.
doi: 10.1021/acs.analchem.5b04578
21 Ruff L. E. ; Marciniak J. Y. ; Sanchez A. B. ; Esener S. C. ; Messmer B. T. Nanotechnol. Rev. 2014, 3, 569.
doi: 10.1515/ntrev-2014-0010
22 Liu D. Y. ; Daubendiek S. L. ; Zillman M. A. ; Ryan K. ; Kool E. T. J. Am. Chem. Soc. 1996, 118 (7), 1587.
doi: 10.1021/ja952786k
23 Zhu G. Z. ; Hu R. ; Zhao Z. L. ; Chen Z. ; Zhang X. B. ; Tan W. H. J. Am. Chem. Soc. 2013, 135 (44), 16438.
doi: 10.1021/ja406115e
24 Lieberman K. R. ; Cherf G. M. ; Doody M. J. ; Olasagasti F. ; Kolodji Y. ; Akeson M. J. Am. Chem. Soc. 2010, 132 (50), 17961.
doi: 10.1021/ja1087612
25 Lu J. ; Chen Y. H. ; Liu D. M. ; Ren W. ; Lu Y. Q. ; Shi Y. ; Piper J. ; Paulsen I. ; Jin D. Y. Anal. Chem. 2015, 87, 10406.
doi: 10.1021/acs.analchem.5b02523
26 Chen X. Q. ; Ye Y. ; Cheng H. ; Jiang Y. W. ; Wu Y. J. Agric. Food Chem. 2009, 57, 5795.
doi: 10.1021/jf9007696
27 Bajaj A. ; Kondaiah P. ; Bhattacharya S. Biomacromolecules 2008, 9, 991.
doi: 10.1021/bm700930y
28 Mohsen M. G. ; Kool E. T. Acc. Chem. Res. 2016, 49 (11), 2540.
doi: 10.1021/acs.accounts.6b00417
29 Lin C. X. ; Wang X. ; Liu Y. ; Seeman N. C. ; Yan H. J. Am. Chem. Soc. 2007, 129, 14475.
doi: 10.1021/ja0760980
[1] 白华荣,范换换,张晓兵,陈卓,谭蔚泓. 核酸适体-纳米材料复合物用于癌症的诊断与靶向治疗研究进展[J]. 物理化学学报, 2018, 34(4): 348-360.
[2] 于泽,李晓宏,李运超,叶明富. 钾离子浓度依赖的铅离子稳定G-四链体构型转化[J]. 物理化学学报, 2018, 34(11): 1293-1298.
[3] 阮弋帆,张楠,朱圆城,赵伟伟,徐静娟,陈洪渊. 光电化学生物分析研究进展[J]. 物理化学学报, 2017, 33(3): 476-485.
[4] 曹国进, 郑卫军. 核酸碱基互变异构体的结构、稳定性及其物理化学性质[J]. 物理化学学报, 2013, 29(10): 2135-2147.
[5] 丛湧, 薛英. 基于机器学习方法的丙型肝炎病毒聚合酶NS5B非核苷抑制剂的定量构效关系研究[J]. 物理化学学报, 2013, 29(08): 1639-1647.
[6] 支泽勇, 刘鹏程, 黄岩谊, 赵新生. 用于单分子动力学实验的微流控混合器[J]. 物理化学学报, 2011, 27(08): 1990-1995.
[7] 康雪丽, 陈启斌, 尚亚卓, 刘洪来. Gemini表面活性剂与DNA在气/液界面上的相互作用[J]. 物理化学学报, 2011, 27(06): 1467-1473.
[8] 李敏杰, 刘卫霞, 彭淳容, 陆文聪. 第一性原理方法预测水相核酸碱基及其代谢物的氧化还原电动势[J]. 物理化学学报, 2011, 27(03): 595-603.
[9] 郜洪文;訾言勤;李玉成. 多色蓝在核酸分子上的Langmuir聚集吸附[J]. 物理化学学报, 2002, 18(06): 540-544.
[10] 黄文,李晓峰,顾惕人. 核糖核酸酶A在DAB-环乙烷溶液中的活性和构象[J]. 物理化学学报, 1996, 12(04): 353-356.
[11] 黄文,李晓峰,顾剔人. 核糖核酸酶在DAB-环已烷反胶束溶液中的活性[J]. 物理化学学报, 1995, 11(07): 579-582.