Automated system for polynucleotide synthesis and purification

• US 6,175,006 B1
• Filed: 03/30/1999
• Issued: 01/16/2001
• Est. Priority Date: 12/21/1988
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Zon, Gerald, Andrus, William A., McCollum, Christie D.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/281,205
• Time in Patent Office: 658 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.33, 536/25.34, 536/25.4, 536/25.41
• US Class Current: 536/25.4
• 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 2219/00351 Means for dispensing and evacuation of reagents
• 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 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.

Claims

1. A method for purifying a polynucleotide comprising

(a) forming a polynucleotide on a solid support by repeated steps of (i) deblocking a 5′

-hydroxyl group of a 5′

-blocked protected nucleoside having a 3′

-hydroxyl and a 5′

-hydroxyl, (ii) reacting the deblocked 5′

-hydroxyl with a 5′

-blocked protected nucleoside monomer selected from the group consisting of 5′

-blocked protected nucleoside-3′

-phosphoramidite and 5′

-blocked protected nucleoside-3′

-hydrogen phosphonate to form either a protected correct-sequence polynucleotide chain or a failure sequence, the failure sequence having a 5′

-hydroxyl, and (iii) repeating steps (i) and (ii) until a desired polynucleotide is obtained, wherein the solid support comprises a chamber containing (1) a nonswellable porous polystyrene support capable of remaining substantially mechanically rigid during polynucleotide synthesis, and (2) a hydrophobic adsorbent capable of non-covalently adsorbing a tritylated polynucleotide,

(b) cleaving the polynucleotide from the nonswellable porous polystyrene support to form a cleavage mixture,

(c) purifying the cleaved polynucleotide from the cleavage mixture by adsorbing the cleaved polynucleotide to the hydrophobic adsorbent and washing the hydrophobic adsorbent to selectively remove unblocked polynucleotides which the cleaved polynucleotide, and

(d) deblocking the 5′

-hydroxyl of the adsorbed polynucleotide to form a deblocked polynucleotide and eluting the deblocked polynucleotide from the hydrophobic adsorbent.

2. The method of claim 1 further including the step of capping said failure sequence by reacting a capping agent with said 5′

-hydroxyl of said failure sequence.

3. The method of claim 2 wherein said 5′

-blocked protected nucleoside monomer is a 5′

-blocked protected nucleoside-3′

-hydrogen phosphonate, and wherein said capping occurs after said reacting.

4. The method of claim 2 wherein said 5′

-blocked protected nucleoside monomer is a 5′

-blocked protected nucleoside-3′

-phosphoramidite, and wherein said capping occurs after said reacting.

5. The method of claim 4 wherein said 5′

-blocked protected nucleoside-3′

-phosphoramidite is defined by the formula;

wherein R₁ is hydrogen or lower alkyl, R₂ is hydrogen, lower alkyl, lower alkoxy, lower aryloxy, or cyano-, nitro-, or sulfono-substituted lower aryloxy, R′

is methyl or beta-cyanoethyl, DMT is dimethoxytrityl, and B is adenine, guanine, thymidine, or cytidine.

6. The method of claim 5 wherein R₁ is hydrogen, ethyl, or propyl, and R₂ is hydrogen, methyl, ethyl, propyl, methoxy, ethoxy, isopropoxy, tert-butyloxy, phenoxy, napthoxy, or biphenoxy.

7. The method of claim 6 wherein R₁ is hydrogen or methyl, and R₂ is hydrogen, methyl, ethoxy, or phenoxy.

8. The method of claim 1 wherein said step of reacting further includes exposing said 5′

-hydroxyl of said correct-sequence chain to said 5′

-blocked protected nucleoside monomer, said 5′

-blocked protected nucleoside monomer being in less than or equal to ten molar excess of said 5′

-hydroxyl of said correct-sequence chain.

9. The method of claim 8 wherein said 5′

-blocked protected nucleoside monomer is a 5′

-blocked protected nucleoside-3′

-phosphoramidite.

10. The method of claim 1, wherein said hydrophobic adsorbent is a nonswellable porous polystyrene adsorbent.

11. The method of claim 1 wherein said 5′

-blocked protected nucleoside monomer is a 5′

-blocked protected nucleoside-3′

-hydrogen phosphonate.

12. The method of claim 1 wherein said 5′

-blocked protected nucleoside monomer is a 5′

-blocked protected nucleoside-3′

-phosphorarnidite.