Nucleic acid assembly optimization using clamped mismatch binding proteins

• US 20070231805A1
• Filed: 03/31/2006
• Published: 10/04/2007
• Est. Priority Date: 03/31/2006
• Status: Abandoned Application

• Current Assignee: Codon Devices Incorporated
• Original Assignee: Codon Devices Incorporated
• Inventors: Baynes, Brian, Blake, William
• Application Number:
  US11/394,708
• Publication Number:
  US 20070231805A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein

Abstract

Certain aspects of the present invention provide methods for increasing the fidelity of multiplex nucleic acid assembly reactions using one or more mismatch binding proteins. Aspects of the invention also provide kits, compositions, devices, and systems for enriching nucleic acid samples for nucleic acids containing correct sequences using clamped forms of one or more mismatch binding proteins.

Claims

1. A method of preparing a nucleic acid having a predetermined sequence, the method comprising:

assembling a sample of double-stranded target nucleic acids from a plurality of different starting nucleic acids in a multiplex assembly reaction;

denaturing and reannealing the sample of double stranded target nucleic acids, thereby forming a heterogeneous pool of reannealed double-stranded heteroduplex and homoduplex nucleic acids if the sample contains a mixture of error-free and error-containing target nucleic acid molecules;

contacting the reannealed double-stranded nucleic acids with a MutS or MutS homolog in the presence of ADP for a time and under conditions that allow the MutS or MutS homolog to bind to heteroduplex nucleic acids,

increasing ATP concentration, after the reannealed double-stranded nucleic acids are circularized, to an ATP concentration that promotes the formation of a clamped form of the MutS or MutS homolog, and

enriching for double-stranded homoduplex nucleic acids by selectively removing circularized double-stranded heteroduplex nucleic acids bound to the MutS or MutS homolog.

2. The method of claim 1, wherein the ATP concentration is greater than the concentration of ADP in the sample.

3. The method of claim 1, wherein the ATP concentration is 40 times greater than the concentration of ADP.

4. The method of claim 1, wherein the ATP concentration is increase to at least 10 micromolar, at least 400 micromolar, or at least 1 mM.

5. The method of claim 1, wherein the double-stranded nucleic acids are double-stranded oligonucleotides.

6. The method of claim 1, wherein heteroduplex nucleic acids are removed by cleaving the nucleic acids bound to the MutS or MutS homolog.

7. The method of claim 1, wherein heteroduplex nucleic acids are removed by filtering the sample through a nitrocellulose filter.

8. The method of claim 1, wherein the double-stranded nucleic acids are linear and are blocked at each end.

9. The method of claim 1, wherein the double-stranded nucleic acids are circular.

10. The method of claim 1, wherein the double-stranded nucleic acids are nucleic acids assembled in a multiplex assembly reaction.

11. The method of claim 1, wherein the double-stranded nucleic acids are circularized by ligating into a vector.

12. The method of claim 1, wherein the double-stranded nucleic acids are circularized before increasing the ATP concentration.

13. The method of claim 1, wherein the double-stranded nucleic acids are circularized before the sample is contacted with the MutS or MutS homolog.

14. The method of claim 1, wherein the ratio of unbound to bound nucleic acids is increased by selectively amplifying double-stranded nucleic acids that are not bound to the MutS or MutS homolog.

15. The method of claim 1, comprising transforming cells with double-stranded nucleic acids not bound to the MutS or MutS homolog.

16. The method of claim 11, further comprising linearizing double-stranded nucleic acids in the sample after removing hetero-duplex nucleic acids bound to the MutS or MutS homolog.

17. The method of claim 1, wherein the double-stranded nucleic acids are 100 to 800 bases in length.

18. The method of claim 1, wherein the enriched homoduplex nucleic acids are assembled to form larger synthetic nucleic acids.

21. The method of claim 1, wherein the MutS homolog is a human MutS homolog, a murine MutS homolog, a rat MutS homolog, a Drosophila MutS homolog, a yeast MutS homolog, or a Saccharomyces cerevisiae MutS homolog.

22. A method of propagating a synthetic nucleic acid, the method comprising:

obtaining a synthetic nucleic acid that was prepared in a method according to claim 1,

transforming a host cell with the synthetic nucleic acid, and

growing the transformed host cell.

23. A method of isolating a polypeptide, the method comprising:

obtaining a host cell transformed with a synthetic nucleic acid that was produced using a nucleic acid enrichment method of claim 1, and

isolating a polypeptide encoded by the synthetic nucleic acid and expressed in the host cell.

24. A method of obtaining a synthetic nucleic acid, the method comprising:

sending sequence information for a target synthetic nucleic acid to a remote site, and

sending delivery information for the target synthetic nucleic acid, wherein the target synthetic nucleic acid is produced at the remote site using a nucleic acid enrichment method of claim 1.

25. A system for producing a synthetic nucleic acid, the system comprising:

a means for producing a sample comprising double-stranded nucleic acids,

a means for exposing the sample to a MutS or MutS homolog under conditions wherein a clamped form of the MutS or MutS homolog is bound to heteroduplex nucleic acids in the sample, and

a means for separating heteroduplex nucleic acids bound to the MutS or MutS homolog from homoduplex nucleic acids not bound to the MutS or MutS homolog using a nucleic acid enrichment method of claim 1.

19-20. -20. (canceled)