Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (1-2): 133-139    DOI: 10.13523/j.cb.1905018
综述     
基因克隆及组装技术的研究进展 *
盛晓菁,齐晓雪,徐蕾,戚智青(),刁勇()
华侨大学医学院分子药物教育部工程研究中心 泉州 362021
The Research Progress of Gene Cloning and Assembly
SHENG Xiao-jing,QI Xiao-xue,XU Lei,QI Zhi-qing(),DIAO Yong()
Insititute of Molecular Medicine, School of MEDIcine, Huaqiao University, Quanzhou 362021, China
 全文: PDF(465 KB)   HTML
摘要：

随着测序技术的发展,已知的DNA序列数量呈指数性增加,为了能更快的探索其未知的生物功能,一些简化组装流程的DNA克隆及组装新技术争相发展起来。其中大部分需要在菌体外构建重组体,但重组酶纯化过程复杂,运送和保存方法要求严格,致使成本较高。最近研究者开发了一些在菌体内进行DNA组装的简易、低成本的新方法。主要对各类基因克隆及组装方法的研究现状、原理和优缺点等进行综述,并结合实际的工作内容展望了未来的发展趋势,希望能为进一步研究开发新技术提供参考。
关键词： DNA重组;  重组酶;  体内重组;  体外重组    
Abstract:

With the development of sequencing technology, the number of known DNA sequences increase exponentially. In order to explore their biological functions more quickly, some new techniques have been developed. Most of these techniques require to construct plasmids in vitro, but the purification process of recombinant enzymes is complicated, transport and preservation are very difficult, which leads to high cost. Recently, researchers have developed simple and low-cost ways to assemble DNA in vivo. The research status, principle, advantages and disadvantages of all kinds of methods are reviewed and the trend is prospected in combination with practical work, hoping to provide a reference for the further research.
Key words: DNA recombination    Recombinase    In vivo recombination    In vitro recombination
收稿日期: 2019-05-11 出版日期: 2020-03-27
ZTFLH:  Q78  
基金资助: * 福建省科技计划高校产学合作项目(2018Y4009);* 福建省科技计划高校产学合作项目(2016N5007)
通讯作者: 戚智青,刁勇     E-mail: zqqi@hotmail.com;diaoyong@hqu.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　　
盛晓菁
齐晓雪
徐蕾
戚智青
刁勇

引用本文:

盛晓菁,齐晓雪,徐蕾,戚智青,刁勇. 基因克隆及组装技术的研究进展 *[J]. 中国生物工程杂志, 2020, 40(1-2): 133-139.

SHENG Xiao-jing,QI Xiao-xue,XU Lei,QI Zhi-qing,DIAO Yong. The Research Progress of Gene Cloning and Assembly. China Biotechnology, 2020, 40(1-2): 133-139.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1905018    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I1-2/133

Methods Principle Specific
site		 Homologous
sequence		 Scar Multiple
fragments		 Time Cost Fidelity Efficiency DNA大小
限制性内切酶 酶切连接 Yes No Yes No >1 week High Normal Low Short
体外重组 Gateway SSR Yes Yes Yes Yes >4d High Normal Normal Normal
RecET HR No Yes No Yes <2d High Normal Normal Normal
SLiCE Not clear No Yes No Yes <2d Low Normal Normal Short
CATCH HR No Yes No Yes <2d High Normal Normal Longer
ExoCET HR Yes Yes No Yes <2d High Normal Normal Longer
体内重组 Red/ET HR No Yes No Yes <2d High High Normal Long
DH5α Not clear No Yes No Yes <2d Low High Low Short
优化的DH5α Not clear No Yes No Yes <2d Low High Normal Short
表1  各种克隆及组装技术的比较
[1] Liu C J, Jiang H, Wu L , et al. OEPR cloning: an efficient and seamless cloning strategy for large- and multi-fragments. Sci Rep, 2017,7:44648.
[2] Trehan A, Kielbus M, Czapinski J , et al. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering. Sci Rep, 2016,6:19121.
[3] Garcia-Nafria J, Watson J F, Greger I H . IVA cloning: A single-tube universal cloning system exploiting bacterial in vivo assembly. Sci Rep, 2016,6:27459.
[4] Beyer H M, Gonschorek P, Samodelov S L , et al. AQUA cloning: a versatile and simple enzyme-free cloning approach. PLoS One, 2015,10(9):e137652.
[5] Koskela E V, Frey A D . Homologous recombinatorial cloning without the creation of single-stranded ends: exonuclease and ligation-independent cloning (ELIC). Mol Biotechnol, 2015,57(3):233-240.
[6] Cao P, Wang L, Zhou G , et al. Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus. Plasmid, 2014,76:40-46.
[7] Oliner J D, Kinzler K W, Vogelstein B . In vivo cloning of PCR products in E. coli. Nucleic Acids Res, 1993,21(22):5192-5197.
[8] Bubeck P, Winkler M, Bautsch W . Rapid cloning by homologous recombination in vivo. Nucleic Acids Res, 1993,21(15):3601-3602.
[9] Kostylev M, Otwell A E, Richardson R E , et al. Cloning should be simple: Escherichia coli DH5alpha-mediated assembly of multiple DNA fragments with short end homologies. PLoS One, 2015,10(9):e137466.
[10] Jacobus A P, Gross J . Optimal cloning of PCR fragments by homologous recombination in Escherichia coli. PLoS One, 2015,10(3):e119221.
[11] Hartley J L, Temple G F, Brasch M A . DNA cloning using in vitro site-specific recombination. Genome Res, 2000,10(11):1788-1795.
[12] Zhang Y, Werling U, Edelmann W . SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res, 2012,40(8):e55.
[13] Zhang Y, Buchholz F, Muyrers J P , et al. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet, 1998,20(2):123-128.
[14] Wang H, Li Z, Jia R , et al. ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. Nucleic Acids Research, 2018,46(5):e28.
[15] Jiang W, Zhao X, Gabrieli T , et al. Cas9-assisted targeting of chromosome segments CATCH enables one-step targeted cloning of large gene clusters. Nat Commun, 2015,6:8101.
[16] Bland M J, Ducos-Galand M, Val M E , et al. An att site-based recombination reporter system for genome engineering and synthetic DNA assembly. BMC Biotechnol, 2017,17(1):62.
[17] Reece-Hoyes J S, Walhout A . Gateway recombinational cloning. Cold Spring Harb Protoc, 2018,1:94912.
[18] Weller S K, Sawitzke J A . Recombination promoted by DNA viruses: phage lambda to herpes simplex virus. Annu Rev Microbiol, 2014,68(1):237-258.
[19] Ivanov E L, Sugawara N, Fishman-Lobell J , et al. Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae. Genetics, 1996,142(3):693-704.
[20] Zhang Y, Werling U, Edelmann W . Seamless ligation cloning extract (SLiCE) cloning method. Methods Mol Biol, 2014,1116:235-244.
[21] Motohashi K . A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis. BMC Biotechnol, 2015,15:47.
[22] Okegawa Y, Motohashi K . A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit. Biochem Biophys Rep, 2015,4:148-151.
[23] Motohashi K . Seamless ligation cloning extract (SLiCE) method using cell lysates from laboratory Escherichia coli strains and its application to SLiP site-directed mutagenesis. Methods Mol Biol, 2017,1498:349-357.
[24] Messerschmidt K, Hochrein L, Dehm D , et al. Characterizing seamless ligation cloning extract for synthetic biological applications. Anal Biochem, 2016,509:24-32.
[25] Garneau J E, Dupuis M E, Villion M , et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010,468(7320):67-71.
[26] Gabrieli T, Sharim H, Fridman D , et al. Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH). Nucleic Acids Res, 2018,46(14):e87.
[27] Sternberg S H, Redding S, Jinek M , et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 2014,507(7490):62-67.
[28] Lovett S T, Hurley R L, Sutera V J , et al. Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways. Genetics, 2002,160(3):851-859.
[29] Muyrers J P, Zhang Y, Buchholz F , et al. RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev, 2000,14(15):1971-1982.
[30] Lu D, Danilowicz C, Tashjian T F , et al. Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology. J Biol Chem, 2019,294(21):8606-8616.
[31] Fu J, Bian X, Hu S , et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nature Biotechnology, 2012,30(5):440-446.
[32] Wang H, Li Z, Jia R , et al. RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nature Protocols, 2016,11(7):1175-1190.
[33] Ma Z, Wang P G . RecET Direct cloning of polysaccharide gene cluster from gram-negative bacteria. Methods Mol Biol, 2019,1954:15-23.
[34] Benoit R M, Ostermeier C, Geiser M , et al. Seamless insert-plasmid assembly at high efficiency and low cost. PLoS One, 2016,11(4):e153158.
[35] Motohashi K . Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods. Biochem Biophys Rep, 2017,9:310-315.
[36] Huang F, Spangler J R, Huang A Y . In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR. PLoS One, 2017,12(8):e183974.
[37] 华侨大学. 一种利用DNA非特异性结合蛋白HU蛋白构建克隆载体的方法:中国(福建), CN201510602398.0. 2019-02-22.[2019-12-15]. http://www.cnipa.gov.cn/.
Qi Z Q, Qi X X, Hou D , et al. A method of constructing cloning vector using DNA nonspecific binding protein HU. Chinese: CN105200074B. 2019-02-22.[2019-12-15]. http://www.cnipa.gov.cn/.
[38] Ohtsuka M, Kimura M, Tanaka M , et al. Recombinant DNA technologies for construction of precisely designed transgene constructs. Curr Pharm Biotechnol, 2009,10(2):244-251.
[39] Okegawa Y, Motohashi K . Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain. Anal Biochem, 2015,486:51-53.
[40] Wang B, Hu Q, Zhang Y , et al. A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum. Microb Cell Fact, 2018,17(1):63.
[41] Taylor G M, Mordaka P M, Heap J T . Start-stop assembly: a functionally scarless DNA assembly system optimized for metabolic engineering. Nucleic Acids Res, 2018,47(3):e17.
[42] Lin D, O’Callaghan C A . MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme. Nucleic Acids Res, 2018,46(19):e113.
[43] van Dolleweerd C J, Kessans S A, an de Bittner K C , et al. MIDAS: A modular DNA assembly system for synthetic biology. ACS Synth Biol, 2018,7(4):1018-1029.
[44] Ding S, Gu Z, Yan R , et al. A novel mode of DNA assembly at electrode and its application to protein quantification. Anal Chim Acta, 2018,1029:24-29.
[45] Casini A, Storch M, Baldwin G S , et al. Bricks and blueprints: methods and standards for DNA assembly. Nat Rev Mol Cell Biol, 2015,16(9):568-576.
[1] 郭曼曼,田开仁,乔建军,李艳妮. 噬菌体重组酶系统在合成生物学中的应用*[J]. 中国生物工程杂志, 2021, 41(8): 90-102.
[2] 马巧妮,王萌,朱兴全. 重组酶介导扩增技术及其在病原微生物快速检测中的应用进展*[J]. 中国生物工程杂志, 2021, 41(6): 45-49.
[3] 郭胜楠, 李信晓, 王峰, 刘昆梅, 丁娜, 扈启宽, 孙涛. 海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 *[J]. 中国生物工程杂志, 2020, 40(3): 9-20.
[4] 朱莹, 倪孟祥, 方宏清. 体内直接克隆大片段DNA的研究进展[J]. 中国生物工程杂志, 2014, 34(4): 95-100.
[5] 叶湘漓, 李大力. GPR126条件性基因敲除载体的快速构建[J]. 中国生物工程杂志, 2013, 33(4): 106-113.
[6] 梁龙, 杨华, 杨永林, 刘守仁, 皮文辉. “Golden Gate”克隆法构建靶向载体[J]. 中国生物工程杂志, 2013, 33(3): 111-116.
[7] 樊祥宇, 谢建平. 分枝杆菌噬菌体重组系统及其应用[J]. 中国生物工程杂志, 2012, 32(09): 101-106.
[8] 姜丽, 叶湘漓, 李大力. 利用Red重组系统快速构建基因打靶载体[J]. 中国生物工程杂志, 2011, 31(10): 88-94.
[9] 吕蓓, 程海荣, 严庆丰, 黄震巨, 李轶女, 罗达, 沈桂芳, 张志芳, 邓子新, 林敏, 程奇. 体外核酸快速扩增技术的发展和不断创新[J]. 中国生物工程杂志, 2011, 31(03): 91-96.
[10] 熊盛,钱垂文,黄立,刘秋英,张美英,王一飞. 50L规模中试发酵重组核苷二磷酸激酶A工程菌[J]. 中国生物工程杂志, 2006, 26(10): 1-6.
[11] 易卓林. DNA改组与分子进化[J]. 中国生物工程杂志, 2006, 26(0): 153-156.
[12] 王佃亮. 重组人溶菌酶研究进展[J]. 中国生物工程杂志, 2003, 23(9): 59-63.
[13] 贾向东, 陈德富, 陈喜文, 郭少影. 几种定向进化技术的比较及文库构建策略[J]. 中国生物工程杂志, 2003, 23(12): 68-72.
[14] 李涛, 卢圣栋. 时空特异性基因打靶的应用及有关进展[J]. 中国生物工程杂志, 2002, 22(2): 56-60.
[15] 王凡强, 马美荣, 王正祥, 诸葛健. 枯草杆菌蛋白酶基因工程的研究进展[J]. 中国生物工程杂志, 2000, 20(2): 41-44.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会