Michael C. Pirrung, Lara Fallon, J.-C. Bradley, William P. Hawe, and Shin Han, P. M.
Gross Chemical Laboratory, Department of Chemistry, Box 90346, Duke University, Durham, NC 27708 and Glenn McGall, Affymetrix, 3380 Central Expressway, Santa Clara, CA 95051
This project is aimed at developing reliable, high-quality chemical synthetic methods to prepare high-density arrays containing thousands of short DNA sequences with either the 5' or 3' end free. Such arrays can be used for hybridization or primer extension reactions. Arrays consisting of complete sets of DNA of a given length can be prepared by the novel technique of light-directed synthesis, and photoremovable groups are key to this method. We have developed a superior new photoremovable group for light-directed DNA synthesis that gives a byproduct that is chemically inert and readily measured by optical and fluorescence methods, permitting the yield in each of the photochemical deprotection steps to be verified. The 3',5'-dimethoxybenzoin (DMB) protecting group is used for the phosphotriester method of DNA synthesis. The 3', 5'-dimethoxybenzoincarbonate (DMB-carbonate) photoremovable protecting group is used for the phosphoramidite method of DNA synthesis. Complete sets of the four protected (allyl, allyloxycarbonyl) nucleoside monomers in which the DMB carbonate is on the 5'-oxygen and the phosphoramidite is on the 3'-oxygen (and vice versa) have been prepared. The former have been used in light-directed synthesis of arrays to both prove the chemistry and test hybridization conditions. The DMB-carbonate method has also been applied to phosphoramidite synthesis of DNA tethered to the solid phase through its 5' end. Work with a collaborating group (J. Shumaker, Baylor) has shown that arrays of DNA with this design can be effectively used for single nucleotide terminating primer extensions (using DNA polymerase and ddNTPs) templated by analyte DNA, permitting comparison sequencing. Finally, this method has been applied to mRNA templates using reverse transcriptase, permitting the analysis of gene expression.
Publications
Michael C. Pirrung, Lara Fallon, Steven W. Shuey, and David C. Lever, "Inverse Phosphotriester DNA Synthesis Using Photochemically-removable Dimethoxybenzoinphosphate Protecting Groups," J. Org. Chem., 60, 0000 (1995).
Michael C. Pirrung and Jean-Claude Bradley, "Comparison of Methods for Photochemical Phosphoramidite-based DNA Synthesis," J. Org. Chem., 60, 6270 (1995
Michael C. Pirrung and Jean-Claude Bradley, "Dimethoxybenzoin Carbonates: Photochemically-removable Alcohol Protecting Groups Suitable for Phosphoramidite-based DNA Synthesis," J. Org. Chem., 60, 1116 (1995).
Michael C. Pirrung and Steve W. Shuey, "Photoremovable Protecting Groups for Phosphorylation of Chiral Alcohols. Asymmetric Synthesis of Phosphotriesters of (-)-3',5'-Dimethoxybenzoin," J. Org. Chem., 59, 3890 (1994).