The Perkin-Elmer Corporation

Applied Biosystems, LLC

William A Andrus

Gerald Zon

Christie D McCollum

Khairuzzaman Bashar Mullah

Lincoln J McBride

J William Efcavitch

A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

Automated system for polynucleotide synthesis and purification

The compounds of the invention are exemplified by the class of diglycolate synthesis supports particularly useful as support reagents for the direct synthesis of 3'-labeled polynucleotides. Generally, the compounds of the invention have the structure ##STR1## where T is an acid-cleavable hydroxyl protecting group, e.g., 4,4'-dimethoxytrityl; L.sub.1 is a linker for connecting a 3'-terminal nitrogen to carbon; L.sub.2 and L.sub.3 are linkers for connecting oxygen and carbon; W is a solid support, e.g., CPG or polystyrene; L.sub.4 is a linker for connecting the solid support to nitrogen; R.sub.1 and R.sub.2 are nitrogen substituents, e.g., hydrogen, lower alkyl, nitrogen protecting group, or label; and R.sub.3 through R.sub.7 are carbon substituents, e.g., hydrogen or lower alkyl. In a first particularly preferred embodiment, the synthesis supports of the invention are exemplified by compounds having the structure ##STR2## where DMT is 4,4'-dimethoxytrityl and W is polystyrene. In a second particularly preferred embodiment, the synthesis supports of the invention are exemplified by compounds having the structure ##STR3## where DMT and W are defined above.

Solid support reagents for the direct synthesis of 3'-labeled

A method and system for polynucleotide synthesis and purification are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure squences caused by chain growth from extraneous support sites. Consequently, currently used rapid purification techniques depending on trityl hydrophobicity give a more highly purified product. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

A method is provided for capping failure sequences in oligonucleotide synthesis by phosphitylation. A phosphite monoester is reacted with the 5' or 3' hydroxyl of the failure sequence between successive condensation steps in a synthesis procedure to form a 5' or 3' phosphite diester with the failure sequence. The phosphite diester substituent is inert with respect to subsequent reaction steps in the synthesis of the desired oligonucleotide product.

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William A Andrus