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

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