Proofreading, error deletion, and ligation method for synthesis of high-fidelity polynucleotide sequences

• US 20030171325A1
• Filed: 01/03/2003
• Published: 09/11/2003
• Est. Priority Date: 01/04/2002
• Status: Abandoned Application

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Becker, Frederick F., Gascoyne, Peter R.C., Vykoukal, Daynene
• Application Number:
  US10/336,610
• Publication Number:
  US 20030171325A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Methods and apparatuses for solid-phase oligonucleotide synthesis and forming long polynucleotides. One exemplary method includes synthesizing a sense oligonucleotide; synthesizing an antisense oligonucleotide; annealing the sense and antisense oligonucleotides to form double stranded DNA (dsDNA); capping the ends of the dsDNA; cleaving the dsDNA wherein cleavage occurs at or near a Watson-Crick base pair mismatch; and digesting uncapped dsDNA. Another exemplary method includes synthesizing a first proofread double stranded DNA (dsDNA); synthesizing a second proofread dsDNA; and ligating the first proofread DNA with the second proofread DNA to form a long polynucleotide.

Claims

1. A method of solid-phase oligonucleotide synthesis comprising:

synthesizing a sense oligonucleotide;

synthesizing an antisense oligonucleotide;

annealing said sense and antisense oligonucleotides to form double stranded DNA (dsDNA);

capping the ends of said dsDNA;

cleaving said dsDNA wherein cleavage occurs at or near a Watson-Crick base pair mismatch; and

digesting uncapped dsDNA.

2. The method of claim 1, further comprising digesting one strand of said dsDNA.

3. The method of claim 1, wherein said oligonucleotide contains 5-100 bases.

4. The method of claim 1, wherein the method occurs on a biochip.

5. The method of claim 4, further comprising using the synthesized oligonucleotide without removing the oligonucleotide from said biochip.

6. The method of claim 1, wherein said solid-phase comprises beads.

7. The method of claim 6, wherein said beads are 2-50 μ

m in diameter.

8. The method of claim 6, wherein said beads comprise dielectrically-engineered beads that are manipulated by dielectrophoresis.

9. The method of claim 6, wherein said beads are gold coated polystyrene beads.

10. The method of claim 6, wherein said beads are coated with a phospholipid.

11. The method of claim 6, wherein said beads are coated with a polyethylene glycol.

12. The method of claim 1, wherein an enzyme is used to cleave said dsDNA.

13. The method of claim 12, wherein said enzyme is an E. Coli endonuclease.

14. The method of claim 12, wherein said enzyme is T7 endonuclease I.

15. The method of claim 1, wherein said dsDNA is cleaved chemically.

16. The method of claim 15, wherein potassium permanganate and hydroxylamine are used to cleave said dsDNA.

17. The method of claim 15, wherein a photoactivated rhodium DNA intercalator is used to cleave said dsDNA.

18. The method of claim 1, wherein a combination of enzymes and/or chemicals are used to cleave said dsDNA.

19. The method of claim 1, further comprising analysis of the DNA with MALDI-TOF MS.

20. The method of claim 1, further comprising using laser assisted deprotection.

21. The method of claim 1, further comprising activating proofreading using laser assisted proofreading activation.

22. The method of claim 1, further comprising control software for the injection and manipulation of fluid droplets on a programmable fluid processor.

23. The method of claim 22, wherein said programmable fluid processor is used for reagent routing and delivery.

24. An apparatus for performing the method of claim 1.

25. A method of forming long polynucleotides comprising:

synthesizing a first proofread double stranded DNA (dsDNA) wherein the synthesis comprises;

synthesizing a sense oligonucleotide;

synthesizing an antisense oligonucleotide;

annealing said sense and antisense oligonucleotides to form dsDNA;

capping the ends of said dsDNA;

cleaving said dsDNA wherein cleaved dsDNA occurs at or near a Watson-Crick base pair mismatch; and

digesting uncapped dsDNA;

synthesizing a second proofread dsDNA; and

ligating said first proofread DNA with said second proofread DNA to form a long polynucleotide.

26. The method of claim 25, wherein 2 -2000 proofread dsDNA are ligated to form said long polynucleotide.

27. The method of claim 26, wherein 10 -500 proofread dsDNA are ligated to form said long polynucleotide.

28. The method of claim 25, wherein the proofread dsDNA are synthesized in parallel.

29. The method of claim 25, wherein the proofread dsDNA are synthesized sequentially.

30. The method of claim 25, wherein ligation occurs using a T4 ligase.

31. The method of claim 25, further comprising digesting one strand of said dsDNA.

32. The method of claim 25, wherein said synthesis and ligation occur on a biochip.

33. The method of claim 32, further comprising a programmable fluidic processor.

34. The method of claim 33, wherein said programmable fluidic processor is used for reagent routing and delivery.

35. The method of claim 31, further comprising using the synthesized oligonucleotide without removing the oligonucleotide from said biochip.

36. An apparatus for performing the method of claim 25.