A rapid and simple transcriptional sequencing method for GC-rich DNA regions.

In genome sequencing project, we encounter the DNA regions that often contain stable secondary structure with high GC content. These regions are difficult to not only amplify by PCR for template preparations, but also deter- mine the DNA sequences using standard Cycle sequencing(CS)method.Tran- scriptional sequencing(TS)is a unique DNA sequencing method using RNA polymerase, and is based on the principles of the chain-termination method, which is a powerful method to analyze GC-rich sequences. In this study, we examined the multiple displacement amplification(MDA)to overcome low ef- ficiency of PCR amplification in GC-rich regions and subjected to TS reaction. Combination of MDA and TS(MDA-TS)was extremely successful with GC content ranging from65% to85%,which are difficult to analyze with PCR and CS. We also report plasmid vector, pTS1,which has the stronger T7and T3 promoters than those of conventional vectors, and the sequence that decreases transcriptional efficiency was removed from its multiple cloning sites. pTS1 resulted in the improved sequencing accuracy and reduced reaction time up to 5min. These results showed that MDA-TS is a rapid and accurate method for the analysis of GC-rich templates.