Transcriptional sequencing: A method for DNA sequencing using RNA polymerase

Abstract:

We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3′-deoxynucleoside triphosphate (3′-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2′-dNTP, which disturbs the Sanger sequencing reaction. An accurate and longer read length was made possible by newly designed four-color dye-3′-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3′-dNTP. This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.

Proceedings of the National Academy of Sciences of the United States of America.1997:95(7):3455

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