METHOD FOR PRODUCING A POPULATION OF OLIGONUCLEOTIDES THAT HAS REDUCED SYNTHESIS ERRORS

• US 20150010953A1
• Filed: 07/03/2013
• Published: 01/08/2015
• Est. Priority Date: 07/03/2013
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sampson, Jeffrey R., Lindstrom, Derek Lee, Ryan, Daniel E.
• Application Number:
  US13/935,201
• Publication Number:
  US 20150010953A1
• Time in Patent Office: Days
• US Class Current: 435/91.52
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

Provided herein is a method for producing a population of oligonucleotides that has reduced synthesis errors. In certain embodiments, the method comprises: a) obtaining an initial population of hairpin oligonucleotide molecules that each comprise a double-stranded stem region and a loop region; b) contacting the double-stranded region of the hairpin oligonucleotide molecules with a mismatch binding protein; and c) eliminating any molecules that bind to the mismatch binding protein, thereby producing a population of oligonucleotides that has reduced synthesis errors. A kit and a composition for performing the method are also provided.

Claims

1. A method for producing a population of oligonucleotides that has reduced synthesis errors, comprising:

a) obtaining an initial population of hairpin oligonucleotide molecules that each comprise a double-stranded stem region and a loop region;

b) contacting the double-stranded region of said hairpin oligonucleotide molecules with a mismatch binding protein; and

c) eliminating any molecules that bind to said mismatch binding protein, thereby producing a population of oligonucleotides that has reduced synthesis errors.

2. The method of claim 1, wherein said method comprises cleaving said loop region from said hairpin oligonucleotide molecules prior to step b).

3. The method of claim 1, wherein said eliminating is done by separating any molecules that bind to said mismatch binding protein from the remainder of the molecules by immobilizing said mismatch binding protein on a solid support.

4. The method of claim 1, wherein the mismatch binding protein is mutS or a variant thereof.

5. The method of claim 1, wherein the mismatch binding protein is T7 endonuclease 1 or a variant thereof.

6. The method of claim 1, wherein said mismatch binding protein is an endonuclease and said eliminating is done by cleaving said double-stranded region at the site of a mismatch.

7. The method of claim 6, wherein said endonuclease also cleaves said loop region.

8. The method of claim 1, further comprising, after the loop region has been cleaved, amplifying a sequence in said double stranded stem region using oligonucleotide primers that bind to the ends of said double stranded region of said hairpin oligonucleotides.

9. The method of claim 1, wherein said initial population of hairpin oligonucleotide molecules comprises multiple different hairpin oligonucleotides molecules that have the same loop region and the same or different double-stranded stem regions.

10. The method of claim 9, wherein the sequences of the double-stranded stem regions of said multiple different hairpin oligonucleotides are at least 80% identical to one another.

11. The method of claim 9, wherein said method further comprises assembling said reduced-error population of oligonucleotides into a plurality of different synthons, wherein said synthons are at least at least 80% identical to one another.

12. The method of claim 11, wherein said assembling is done by polymerase chain assembly (PCA) or ligase chain assembly (LCA).

13. The method of claim 1, wherein said hairpin oligonucleotide molecules comprise a site for a restriction enzyme in said double stranded region, proximal to said loop.

14. The method of claim 1, wherein loop region of said hairpin oligonucleotide molecules is at least four nucleotides in length.

15. The method of claim 1, wherein double-stranded region of said hairpin oligonucleotide molecules is at least 20 nucleotides in length.

16. A kit comprising:

a) a population of hairpin oligonucleotide molecules that each comprise a double-stranded stem region and a loop region; and

b) a mismatch binding protein.

17. The kit of claim 16, wherein the sequences of the double-stranded stem regions of said hairpin oligonucleotide molecules are at least 80% identical to one another.

18. The kit of claim 16, wherein said mismatch binding protein is T7 endonuclease I, mutS or a variant thereof.

19. The kit of claim 16, wherein said hairpin oligonucleotide molecules comprise a site for a restriction enzyme in said double stranded region, proximal to said loop and said kit further comprises said restriction enzyme.

20. A composition comprising:

a) a population of hairpin oligonucleotide molecules that each comprise a double-stranded stem region and a loop region; and

b) a mismatch binding protein;

wherein the mismatch binding protein is bound to any hairpin oligonucleotide molecules that have a synthesis error in the double-stranded stem region.