Automated system for polynucleotide synthesis and purification

• US 5,047,524 A
• Filed: 12/21/1988
• Issued: 09/10/1991
• Est. Priority Date: 12/21/1988
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Zon, Gerald, Andrus, William A., McCollum, Christie D.
• Primary Examiner(s): Brown, Johnnie R.
• Assistant Examiner(s): Kunz, Gary L.
• Application Number:
  US07/287,890
• Time in Patent Office: 993 Days
• Field of Search: 536/27, 536/28, 536/29, 935/88
• US Class Current: 536/25.31
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/0068 Means for controlling the apparatus of the process
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 for creating libraries

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.

Claims

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.

2. The method of claim 1 wherein said step of cleaving includes deprotecting said polynucleotide.

3. 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.

4. The method of claim 3 wherein said hydrophobic adsorbent is a nonswellable polystyrene adsorbent.

5. 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.

6. 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.

7. The method of claim 6 wherein said 5'"'"'-tritylated protected nucleoside-3'"'"'-phosphoramidite is defined by the formula:

##STR5## wherein;

R₁ is selected from the group consisting of hydrogen and lower alkyl;

R₂ 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.

8. The method of claim 7 wherein R₁ is selected from the group consisting of hydrogen, methyl, ethyl, and propyl, and wherein R₂ is selected from the group consisting of hydrogen, methyl, ethyl, propyl, methoxy, ethoxy, isopropoxy, tert-butyloxy, phenoxy, napthoxy, and biphenoxy.

9. The method of claim 8 wherein R₁ is selected from the group consisting of hydrogen and methyl, and wherein R₂ is selected from the group consisting of hydrogen, methyl, ethoxy, and phenoxy.

10. The method of claim 4 wherein said nonswellable polystyrene adsorbent is treated with an alkylammonium salt solution prior to said step of purifying.