Automated system for polynucleotide synthesis and purification
First Claim
1. A method for synthesizing a polynucleotide of a predetermined sequence, the method comprising the steps of:
- (a) providing a nonswellable porous polystyrene support, remaining substantially mechanically rigid during synthesis;
(b) providing a 5'"'"'-tritylated protected nucleoside, the 5'"'"'-tritylated protected nucleoside being attached to the nonswellable porous polystyrene support, such that the 5'"'"'-tritylated protected nucleoside forms a protected correct-sequence chain;
(c) detritylating the correct-sequence chain to free a 5'"'"'-hydroxyl;
(d) reacting with the 5'"'"'-hydroxyl of the correct-sequence chain a 5'"'"'-tritylated protected nucleoside monomer selected from the group consisting of 5'"'"'-tritylated protected nucleoside-3'"'"'-phosphoramidite and 5'"'"'-tritylated protected nucleoside-3'"'"'-hydrogen phosphonate to form either a protected correct-sequence chain or a failure sequence, the failure sequence having a 5'"'"'-hydroxyl;
(e) repeating steps (c) and (d) until the polynucleotide of the predetermined sequence is obtained;
(f) cleaving the polynucleotide from the nonswellable porous polystyrene support to form a cleavage mixture, the cleavage mixture containing tritylated polynucleotide and untritylated failure sequences; and
(g) purifying the polynucleotide by exposing the cleavage mixture to a hydrophobic adsorbent, washing the hydrophobic adsorbent to preferentially remove untritylated failure sequences, detritylating the 5'"'"'-hydroxyl of the tritylated polynucleotide, and eluting the untritylated polynucleotide from the hydrophobic adsorbent.
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Abstract
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.
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Citations
10 Claims
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1. A method for synthesizing a polynucleotide of a predetermined sequence, the method comprising the steps of:
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(a) providing a nonswellable porous polystyrene support, remaining substantially mechanically rigid during synthesis; (b) providing a 5'"'"'-tritylated protected nucleoside, the 5'"'"'-tritylated protected nucleoside being attached to the nonswellable porous polystyrene support, such that the 5'"'"'-tritylated protected nucleoside forms a protected correct-sequence chain; (c) detritylating the correct-sequence chain to free a 5'"'"'-hydroxyl; (d) reacting with the 5'"'"'-hydroxyl of the correct-sequence chain a 5'"'"'-tritylated protected nucleoside monomer selected from the group consisting of 5'"'"'-tritylated protected nucleoside-3'"'"'-phosphoramidite and 5'"'"'-tritylated protected nucleoside-3'"'"'-hydrogen phosphonate to form either a protected correct-sequence chain or a failure sequence, the failure sequence having a 5'"'"'-hydroxyl; (e) repeating steps (c) and (d) until the polynucleotide of the predetermined sequence is obtained; (f) cleaving the polynucleotide from the nonswellable porous polystyrene support to form a cleavage mixture, the cleavage mixture containing tritylated polynucleotide and untritylated failure sequences; and (g) purifying the polynucleotide by exposing the cleavage mixture to a hydrophobic adsorbent, washing the hydrophobic adsorbent to preferentially remove untritylated failure sequences, detritylating the 5'"'"'-hydroxyl of the tritylated polynucleotide, and eluting the untritylated polynucleotide from the hydrophobic adsorbent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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Specification