Efficient amplification of genes involved in microbial secondary metabolism by an improved genome walking method.

作者: Jing Deng , Maochen Wei , Boyang Yu , Yijun Chen

DOI: 10.1007/S00253-010-2569-4

关键词:

摘要: Genome walking is a commonly used technique for the identification of DNA sequences adjacent to known regions. Despite development various genome methods, nonspecific products are often produced in certain circumstances, especially when GC-rich dealt with. To effectively resolve such technical issues, simple nested polymerase chain reaction-based method has been developed by implementing progressively decreased annealing temperature from 70 degrees C 47.5 first round amplification and high 65 second amplification. During entire process, lower ramp rate 1.5 s(-1) cooling 2.5 performed reach temperature. Using this method, we successfully obtained upstream downstream three genes involved biosynthetic pathways secondary metabolites two bacterial genomes. The efficient target longer than Kb with GC content up 75.0% indicates that present could be valuable tool investigation metabolites.

参考文章(31)
Yan Yuanxin, An Chengcai, Li Li, Gu Jiayu, Tan Guihong, Chen Zhangliang, T‐linker‐specific ligation PCR (T‐linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends Nucleic Acids Research. ,vol. 31, ,(2003) , 10.1093/NAR/GNG068
Markus C. Carnio, Torsten Stachelhaus, Kevin P. Francis, Siegfried Scherer, Pyridinyl polythiazole class peptide antibiotic micrococcin P1 , secreted by foodborne Staphylococcus equorum WS2733, is biosynthesized nonribosomally European Journal of Biochemistry. ,vol. 268, pp. 6390- 6401 ,(2001) , 10.1046/J.0014-2956.2001.02591.X
D. W. Udwary, L. Zeigler, R. N. Asolkar, V. Singan, A. Lapidus, W. Fenical, P. R. Jensen, B. S. Moore, Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica Proceedings of the National Academy of Sciences of the United States of America. ,vol. 104, pp. 10376- 10381 ,(2007) , 10.1073/PNAS.0700962104
Andreas Rascher, Zhihao Hu, Nina Viswanathan, Andreas Schirmer, Ralph Reid, William C Nierman, Matthew Lewis, C.Richard Hutchinson, Cloning and characterization of a gene cluster for geldanamycin production inStreptomyces hygroscopicusNRRL 3602 FEMS Microbiology Letters. ,vol. 218, pp. 223- 230 ,(2003) , 10.1016/S0378-1097(02)01148-5
Kyl V. Myrick, William M. Gelbart, Universal Fast Walking for direct and versatile determination of flanking sequence Gene. ,vol. 284, pp. 125- 131 ,(2002) , 10.1016/S0378-1119(02)00384-0
Tony Triglia, M.Gregory Peterson, David J. Kemp, A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences Nucleic Acids Research. ,vol. 16, pp. 8186- 8186 ,(1988) , 10.1093/NAR/16.16.8186
Jay D. Parker, Peter S. Rabinovitch, Glenna C. Burmer, Targeted gene walking polymerase chain reaction Nucleic Acids Research. ,vol. 19, pp. 3055- 3060 ,(1991) , 10.1093/NAR/19.11.3055
G. Pfeifer, S. Steigerwald, P. Mueller, B Wold, A. Riggs, Genomic sequencing and methylation analysis by ligation mediated PCR Science. ,vol. 246, pp. 810- 813 ,(1989) , 10.1126/SCIENCE.2814502
A.S Rishi, Neil D Nelson, Arun Goyal, Genome walking of large fragments: an improved method. Journal of Biotechnology. ,vol. 111, pp. 9- 15 ,(2004) , 10.1016/J.JBIOTEC.2004.03.008