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US5047524A
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Automated system for polynucleotide synthesis and purification
Assignee: Applied Biosystems LLC
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Filing: 1988-12-21
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Granted: 1991-09-10
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Expires: 2008-12-21
Abstract
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.
Inventors
- 👤 William A. Andrus
- 👤 Christie D. McCollum
- 👤 Gerald Zon
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 11:45
CPC Classifications
C; C07; C07H; C07H21/00; B; B01; B01J; B01J19/00; B01J19/0046; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1003; C12N15/1006; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00279; B01J2219/00281; B01J2219/00286; B01J2219/00351; B01J2219/00389; B01J2219/00452; B01J2219/00454; B01J2219/00497; B01J2219/005; B01J2219/00583; B01J2219/0059; B01J2219/00596; B01J2219/0068; B01J2219/00686; B01J2219/00718; B01J2219/0072; B01J2219/00722; C40; C40B; C40B40/00; C40B40/04; C40B40/06; C40B50/00; C40B50/14; C40B60/00; C40B60/14
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
192 words
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.
Claim 2
Independent Claim
192 words
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.
Claim 3
Dependent Claim
14 words
The method of claim 1 wherein said step of cleaving includes deprotecting said polynucleotide.
Claim 4
Dependent Claim
26 words
The method of claim 2 further including the step of capping said failure sequence by reacting a capping agent with said 5'-hydroxyl of said failure sequence.
Claim 5
Dependent Claim
14 words
The method of claim 3 wherein said hydrophobic adsorbent is a nonswellable polystyrene adsorbent.
Claim 6
Dependent Claim
28 words
The method of claim 3 wherein said 5'-tritylated protected nucleoside monomer is 5'-tritylated protected nucleoside-3'-hydrogen phosphonate, and wherein said step of capping occurs after said step of repeating.
Claim 7
Dependent Claim
27 words
The method of claim 3 wherein said 5'-tritylated protected nucleoside monomer is 5'-tritylated protected nucleoside-3'-phosphoramidite, and wherein said step of capping occurs after said step of reacting.
Claim 8
Dependent Claim
80 words
The method of claim 6 wherein said 5'-tritylated protected nucleoside-3'-phosphoramidite is defined by the formula: ##STR5## wherein: R 1 is selected from the group consisting of hydrogen and lower alkyl; R 2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, lower aryloxy, cyano-, nitro-, or sulfono-substituted lower aryloxy; R' is selected from the group consisting of methyl and beta-cyanoethyl; DMT is dimethoxytrityl; and B is selected from the group consisting of adenine, guanine, thymidine, and cytidine.
Claim 9
Dependent Claim
43 words
The method of claim 7 wherein R 1 is selected from the group consisting of hydrogen, methyl, ethyl, and propyl, and wherein R 2 is selected from the group consisting of hydrogen, methyl, ethyl, propyl, methoxy, ethoxy, isopropoxy, tert-butyloxy, phenoxy, napthoxy, and biphenoxy.
Claim 10
Dependent Claim
34 words
The method of claim 8 wherein R 1 is selected from the group consisting of hydrogen and methyl, and wherein R 2 is selected from the group consisting of hydrogen, methyl, ethoxy, and phenoxy.
Claim 11
Dependent Claim
23 words
The method of claim 4 wherein said nonswellable polystyrene adsorbent is treated with an alkylammonium salt solution prior to said step of purifying.
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