Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2009, Vol. 29 Issue (02): 71-75    
技术与方法     
RecA蛋白介导的三链核酸结构形成及其序列提取
方刚1,张社民2
1. 西安文理学院生命科学系
2. 陕西理工学院
RecA-mediated triple-stranded DNA structure formed by 100bp dsDNA and extracted by affinity capture
 全文: PDF(629 KB)   HTML
摘要: 人工合成的单链DNA分子经PCR扩增形成双链DNA分子。将RecA蛋白与生物素标记的寡聚核酸探针序列在ATPγS存在的情况下共同哺育,使RecA蛋白包裹寡聚核酸探针,然后加入含同源序列的上述双链DNA分子经适当环境哺育形成了稳定的局部三链核酸结构。通过加入链亲和素包裹的磁珠吸附生物素化的探针,这样同源双链DNA分子与寡聚核酸探针形成的局部三链核酸结构也被吸附在磁珠上。使用磁分离装置提取这一结构,逐步降低盐离子浓度以洗脱双链DNA分子。将洗脱液中残留的蛋白质去除,经PCR扩增可获得目的DNA序列。同时使用同源探针和非同源探针在其它序列中提取目的DNA序列,结果显示目的DNA序列只被同源探针提取。实验结果显示了这一三链核酸结构形成的序列特异性,并且其稳定性随盐离子浓度降低而下降。提示在这一结构中同源的寡聚核酸单链与双链DNA分子形成了氢键结合,同时提示使用文中描述的方法可以提取特异的序列,用以克隆相应的基因。
关键词: 三链DNA结构;RecA蛋白;同源的;ATPγS    
Abstract:

A sequence of single-stranded DNA molecule was synthesized and PCR amplified. Then sequence-specific recognition of doublestranded DNA by its homologous deoxyoligonucleotides, which were coated with RecA protein, was performed in the presence of the non-hydrolyzable analog of ATP—ATPγS. After the reaction, local stable triplestranded DNA structure was formed. By using affinity capture, the target double-stranded DNA strands were retained together with the biotinylated deoxyoligonucleotide probes and extracted. In the same way, the target DNA sequence can only be extracted by using homologous deoxyoligonucleotides probe. It demonstrates that the recognition is sequence specific. The observations also show that the RecA-mediated triple-stranded DNA structure can be dissociated with the gradient decrease of saline concentration. It is suggested that hydrogen bonds are formed between incoming strand and intrinsic doublestranded DNA and the methods can be used to isolate special DNA sequences conveniently and efficiently.

收稿日期: 2008-03-05 出版日期: 2009-03-31
ZTFLH:  Q523.5  
基金资助:

国家自然科学基金

通讯作者: 方刚   
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  
方刚 张社民

引用本文:

方刚,张社民. RecA蛋白介导的三链核酸结构形成及其序列提取[J]. 中国生物工程杂志, 2009, 29(02): 71-75.

FANG Gang- Zhang-She-Min. RecA-mediated triple-stranded DNA structure formed by 100bp dsDNA and extracted by affinity capture. China Biotechnology, 2009, 29(02): 71-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I02/71

[1] FrankKamenetskii M D, Mirkin S D. Triplex DNA structures. Annu Rev Biochem, 1995, 64: 65~95 [2] Zhurkin V B, Raghunathan G, Ulyanov N B, et al. A parallel DNA triplex as a mode for the intermediate in homologous recombination. J Mol Biol, 1994, 239: 181~200 [3] Richard A J Darby, Matthieu S, Catherine M, et al. High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler. Nucleic Acids Research, 2002, 30(9):e39 [4] Hsieh P, CameriniOtero C S, CameriniOtero R D. Pairing of homologous DNA sequences by proteins: evidence for threestranded DNA. Genes Dev, 1990, 4: 1951~1963 [5] Rao B J, Dutreix M, Radding C M. Stable threestranded DNA made by RecA protein. Proc Natl Acad Sci USA, 1991, 88: 2984~2988 [6] Umlauf S W, Cox M M, Inman R B. Triplehelical DNA pairing intermediates formed by RecA protein. J Biol Chem, 1990, 265: 16898~16912 [7] Muller B, Burdett I, West S C. Unusual stability of recombination intermediates made by Escherichia coli RecA protein. EMBO J, 1992, 7: 2685~2693 [8] Efim I Golub, David C Ward, Charles M Radding. Joints formed by RecA protein from oligonucleotides and duplex DNA block initiation and elongation of transcription. Nucleic Acids Research, 1992, 12 (20): 3121~3125 [9] YanceyWrona J E, CameriniOtero R D. The search for DNA homology does not limit stable homologous pairing promoted by RecA protein. Curr Biol, 1995, 5: 1149~1158 [10] Miya D Drury, Eric B Kmiec. DNA pairing is an important ste Pin the process of targeted nucleotide exchange. Nucleic Acids Research, 2003, 31 (3): 899~910 [11] Huamin J I, Ioydm S, Richard A G. Rapid isolation of cosmid insert DNA by triplehelixmediated affinity capture. Genetic Analysis Tech Application, 1994, 112 (2): 43~47 [12] Shigemori Y, Oishi M. Specific cleavage of DNA molecules at RecAmediated triplestranded structure. Nucleic Acids Research, 2004, 32(1):4. doi: 10.1093/nar/gnh004. Published online January 4.2004 [13] Oleg N Voloshin, R Daniel C. The duplex DNA is very underwound in the threestranded RecA proteinmediated synaptic complex. Genes to Cells, 1997, 2: 303~314 [14] Lu Y, Ding H P, Xu H, et al. Study on preparation of protein A magnetic nanoparticle carrier and its application. China Biotechnology, 2008, 28 (1): 13~17

No related articles found!