Methods for improving the sequence fidelity of synthetic double-stranded oligonucleotides

• US 6,664,112 B2
• Filed: 06/01/2001
• Issued: 12/16/2003
• Est. Priority Date: 06/02/2000
• Status: Expired due to Term

• Current Assignee: Blue Heron Biotech, LLC (Eurofins Scientific SE)
• Original Assignee: Blue Heron Biotechnology Incorporated
• Inventors: Mulligan, John T., Tabone, John C.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Lu, Frank Wei Min
• Application Number:
  US09/872,761
• Publication Number:
  US 20020077471A1
• Time in Patent Office: 928 Days
• Field of Search: 435/6, 435/91.1, 435/183, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 422/68.1, 422/50
• US Class Current: 436/94
• CPC Class Codes: C07H 1/06 Separation; Purification
• 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 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Synthetic oligonucleotides, such as synthetic DNA, often contain sequence errors due to synthetic failures (e.g., side products and/or truncated products). Methods are provided herein for improving the sequence fidelity of synthetic double-stranded oligonucleotides by separative depletion of synthetic failures. Separation is effected by utilization of methodologies in a preparative mode under denaturing conditions. A preferred use of the methods relates to gene synthesis.

Claims

1. A method for improving the sequence fidelity of synthetic double-stranded oligonucleotides, comprising subjecting synthetic double-stranded oligonucleotides to preparative high performance liquid chromatography (HPLC) under partially denaturing conditions sufficient to separate synthetic double-stranded oligonucleotides into two populations of which one population is enriched for synthetic failures and the other population is depleted of synthetic failures.

2. A method according to claim 1, wherein the chromatography is denaturing high performance liquid chromatography (DHPLC).

3. A method according to claim 1 or claim 2, wherein the oligonucleotides comprise synthetic double-stranded DNA.

4. A method according to claim 3, wherein the DNA comprises one or more fragments of a gene.

5. A method according to claim 1 or claim 2, wherein the synthetic failures separated are molecules containing a uridine, apurinic, apyrimidinic or diaminopurine residue.

6. A method according to claim 1 or claim 2, wherein the double-stranded oligonucleotides are synthesized chemically.

7. A method according to claim 6, wherein the oligonucleotides comprise double-stranded DNA.

8. A method according to claim 7, wherein the DNA comprises one or more fragments of a gene.

9. A method according to claim 3, further comprising joining oligonucleotides from the population depleted of synthetic failures, to other synthetic oligonucleotides.

10. A method according to claim 9, wherein a gene or gene fragment is formed when the oligonucleotides are joined.

11. A method according to claim 7, further comprising joining oligonucleotides from the population depleted of synthetic failures, to other synthetic oligonucleotides.

12. A method according to claim 11, wherein a gene or gene fragment is formed when the oligonucleotides are joined.