3 -strongostrong-modified nucleotides as reversible terminators for.pdfVIP

3-O-modified nucleotides as reversible terminators for pyrosequencing †‡ †‡ †‡ † † † Jian Wu* , Shenglong Zhang* , Qinglin Meng* , Huanyan Cao* , Zengmin Li* , Xiaoxu Li* , Shundi Shi*, Dae Hyun Kim*§ † †‡¶ †¶ , Lanrong Bi* , Nicholas J. Turro , and Jingyue Ju* *Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032; and Departments of †Chemical Engineering, ‡Chemistry, and §Biomedical Engineering, Columbia University, New York, NY 10027 Contributed by Nicholas J. Turro, August 10, 2007 (sent for review July 3, 2007) Pyrosequencing is a method used to sequence DNA by detecting released. The PPi is used by ATP sulfurylase to convert aden- the pyrophosphate (PPi) group that is generated when a nucleotide osine 5-phosphosulfate to ATP, which provides the energy to is incorporated into the growing DNA strand in polymerase reac- the luciferase-mediated conversion of luciferin to oxyluciferin, tion. However, this method has an inherent difficulty in accurately which generates visible light. If the added nucleotide is not deciphering the homopolymeric regions of the DNA templates. We incorporated, no light will be produced and the nucleotide will report here the development of a method to solve this problem by simply be washed away or degraded by the enzyme apyrase. using nucleotide reversible terminators. These nucleotide ana- Pyrosequencing has been widely used in single nucleotide poly- logues are modified with a reversible chemical moiety capping the morphism detection and DNA methylation analysis (16, 17). 3-OH group to