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

概要

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In genome sequencing project, we encounter the DNA regions that oftencontain stable secondary structure with high GC content. These regions aredifficult to not only amplify by PCR for template preparations, but also determinethe DNA sequences using standard Cycle sequencing（CS）method. Transcriptionalsequencing（TS）is a unique DNA sequencing method using RNApolymerase, and is based on the principles of the chain-termination method,which is a powerful method to analyze GC-rich sequences. In this study, weexamined the multiple displacement amplification（MDA）to overcome low efficiencyof PCR amplification in GC-rich regions and subjected to TS reaction.Combination of MDA and TS（MDA-TS）was extremely successful with GCcontent ranging from 65% to 85%，which are difficult to analyze with PCR andCS. We also report plasmid vector, pTS1，which has the stronger T7and T3promoters than those of conventional vectors, and the sequence that decreasestranscriptional efficiency was removed from its multiple cloning sites. pTS1resulted in the improved sequencing accuracy and reduced reaction time up to５min. These results showed that MDA-TS is a rapid and accurate method forthe analysis of GC-rich templates.
The Graduate School of Veterinary Medicine, Hokkaido Universityの論文
2006-02-28