title: Solid-Phase Purification of Synthetic DNA Sequences.
abstract: Although high-throughput methods for solid-phase synthesis of DNA sequences are currently available for synthetic biology applications and technologies for large-scale production of nucleic acid-based drugs have been exploited for various therapeutic indications, little has been done to develop high-throughput procedures for the purification of synthetic nucleic acid sequences. An efficient process for purification of phosphorothioate and native DNA sequences is described herein. This process consists of functionalizing commercial aminopropylated silica gel with aminooxyalkyl functions to enable capture of DNA sequences carrying a 5'-siloxyl ether linker with a "keto" function through an oximation reaction. Deoxyribonucleoside phosphoramidites functionalized with the 5'-siloxyl ether linker were prepared in yields of 75-83% and incorporated last into the solid-phase assembly of DNA sequences. Capture of nucleobase- and phosphate-deprotected DNA sequences released from the synthesis support is demonstrated to proceed near quantitatively. After shorter than full-length DNA sequences were washed from the capture support, the purified DNA sequences were released from this support upon treatment with tetra-n-butylammonium fluoride in dry DMSO. The purity of released DNA sequences exceeds 98%. The scalability and high-throughput features of the purification process are demonstrated without sacrificing purity of the DNA sequences.
authors.affiliation: Laboratory of Biological Chemistry, Division of Biotechnology Review and Research IV, Center for Drug Evaluation and Research, Food and Drug Administration , 10903 New Hampshire Avenue, Silver Spring, Maryland 20933, United States.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Grajkowski
authors.affiliation: Laboratory of Biological Chemistry, Division of Biotechnology Review and Research IV, Center for Drug Evaluation and Research, Food and Drug Administration , 10903 New Hampshire Avenue, Silver Spring, Maryland 20933, United States.
authors.forename: Jacek
authors.initials: J
authors.lastname: Cieslak
authors.affiliation: Laboratory of Biological Chemistry, Division of Biotechnology Review and Research IV, Center for Drug Evaluation and Research, Food and Drug Administration , 10903 New Hampshire Avenue, Silver Spring, Maryland 20933, United States.
authors.forename: Serge L
authors.initials: SL
authors.lastname: Beaucage
mesh_terms: D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D009696:Nucleic Acids; D009943:Organophosphorus Compounds; D010710:Phosphates / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D012910:Snake Venoms / Q000201:enzymology; D060327:Solid-Phase Synthesis Techniques
chemical_list: D009696:Nucleic Acids; D009943:Organophosphorus Compounds; D010710:Phosphates; D012910:Snake Venoms; C434331:phosphoramidite; D012822:Silicon Dioxide; D004247:DNA; C035638:thiophosphoric acid