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.