Claims
- 1. A method of synthesizing a DNA sequence comprising:
(i) dividing the DNA sequence recursively into small pieces of DNA, wherein adjacent pieces comprise overlapping regions; (ii) optimizing the sequences of the pieces of DNA resulting from each recursive division to strengthen correct hybridizations and to disrupt incorrect hybridizations; (iii) obtaining the optimized small pieces of DNA, wherein the overlapping regions of any adjacent pieces of single-stranded DNA are complementary; (iv) combining the pieces of DNA derived from the division of the next-larger piece of DNA; (v) allowing the pieces of DNA to self-assemble to form a DNA construct comprising single-stranded DNA segments connected by double-stranded overlap regions; (vi) producing the next-larger piece of DNA from the DNA construct; and (vii) repeating steps (iv), (v), and (vi) in reverse order of the recursive division in step (i) to produce the DNA sequence.
- 2. The method of claim 1, wherein a next-larger piece of DNA comprises a mixture of DNA molecules, the method further comprising:
selecting a DNA molecule from the mixture likely to have the correct DNA sequence, and using the selected DNA molecule in the synthesis of the DNA sequence.
- 3. The method of claim 2, wherein a DNA molecule is separated from the mixture by cloning.
- 4. The method of claim 2, wherein the selection comprises sequencing a sample of DNA molecules from the mixture and selecting a DNA molecule with the desired DNA sequence.
- 5. The method of claim 2, wherein the selection comprises expressing a polypeptide from each member of a sample of DNA molecules from the mixture, determining the molecular weight of the polypeptide, and selecting a DNA molecule from which a polypeptide with a predetermined molecular weight is expressed.
- 6. The method of claim 5, wherein a start codon and/or a stop codon is incorporated into the DNA molecule from which a polypeptide is expressed.
- 7. The method of claim 6, wherein the reading frame of the DNA molecule is adjusted with respect to the start codon and/or stop codon.
- 8. The method of claim 5, wherein each member of the sample of DNA molecules is inserted into an expression vector, and wherein the expression vector comprises a stop codon downstream from the inserted DNA molecule.
- 9. The method of claim 5, wherein the molecular weight of the polypeptide is determined by electrophoresis.
- 10. The method of claim 1, wherein the DNA sequence comprises a regulatory sequence.
- 11. The method of claim 1, wherein the DNA sequence comprises an intergenic sequence.
- 12. The method of claim 1, wherein the DNA sequence encodes a polypeptide.
- 13. The method of claim 12, wherein the polypeptide is a full-length protein.
- 14. The method of claim 1, wherein dividing the DNA sequence into small pieces of DNA is performed in a single division.
- 15. The method of claim 1, wherein dividing the DNA sequence into small pieces of DNA is performed in a plurality of divisions.
- 16. The method of claim 15, wherein the DNA sequence is divided into pieces of DNA of about 1,500 bases long or shorter.
- 17. The method of claim 1, wherein the small pieces of DNA are about 60 bases long or shorter.
- 18. The method of claim 17, wherein the small pieces of DNA are about 50 bases long or shorter.
- 19. The method of claim 1, wherein the overlapping regions comprise from about 6 to about 60 base-pairs.
- 20. The method of claim 1, wherein optimizing comprises calculating a melting temperature for the pieces of DNA.
- 21. The method of claim 1, wherein optimizing comprises calculating a parameter related to hybridization propensity for the pieces of DNA.
- 22. The method of claim 21, wherein the parameter is selected from the group consisting of free energy, enthalpy, entropy, and arithmetic or algebraic combinations thereof.
- 23. The method of claim 20, wherein the melting temperature of the lowest melting correct hybridization is at least 1° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 24. The method of claim 20, wherein the melting temperature of the lowest melting correct hybridization is at least 4° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 25. The method of claim 20, wherein the melting temperature of the lowest melting correct hybridization is at least 8° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 26. The method of claim 20, wherein the melting temperature of the lowest melting correct hybridization is at least 16° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 27. The method of claim 1, wherein optimizing comprises taking advantage of the degeneracy in the regulatory region consensus sequence.
- 28. The method of claim 1, wherein optimizing comprises direct base assignment.
- 29. The method of claim 1, wherein optimizing comprises adjusting boundary points between adjacent pieces of DNA.
- 30. The method of claim 1, wherein optimizing comprises permuting silent codon substitutions.
- 31. The method of claim 1, wherein at least one of the optimized small pieces of DNA is synthetic.
- 32. The method of claim 1, wherein at least one of the optimized small pieces of DNA is single-stranded.
- 33. The method of claim 1, wherein a single-stranded DNA segment has a length of from about zero bases to about 20 bases.
- 34. The method of claim 1, wherein the next-larger piece of DNA is produced by cloning the DNA construct.
- 35. The method of claim 34, wherein the cloning is selected from the group consisting of exonuclease III cloning, topoisomerase cloning, restriction enzyme cloning, and homologous recombination cloning.
- 36. The method of claim 1, wherein the next-larger piece of DNA is produced by ligating the DNA construct.
- 37. The method of claim 1, wherein the next-larger piece of DNA is produced by extending the DNA construct by a reaction using DNA polymerase.
- 38. The method of claim 37, wherein the DNA polymerase is a proof-reading DNA polymerase.
- 39. The method of claim 37, further comprising mixing a DNA polymerase primer with the pieces of DNA derived from the division of the next-larger piece of DNA.
- 40. The method of claim 1, further comprising designing a restriction site into an overlapping region.
- 41. The method of claim 40, further comprising digesting the restriction site with a site-specific restriction enzyme.
- 42. A DNA sequence synthesized according to a method comprising:
(i) dividing the DNA sequence recursively into small pieces of DNA, wherein adjacent pieces comprise overlapping regions; (ii) optimizing the sequences of the pieces of DNA resulting from each recursive division to strengthen correct hybridizations and to disrupt incorrect hybridizations; (iii) obtaining the optimized small pieces of DNA, wherein the overlapping regions of any adjacent pieces of single-stranded DNA are complementary; (iv) combining the pieces of DNA derived from the division of the next larger piece of DNA; (v) allowing the pieces of DNA to self-assemble to form a DNA construct comprising single-stranded DNA segments connected by double-stranded overlap regions; (vi) producing the next-larger piece of DNA from the DNA construct; and (vii) repeating steps (iv), (v), and (vi) in reverse order of the recursive division in step (i) to produce the DNA sequence.
- 43. The DNA sequence of claim 42, wherein a next-larger piece of DNA comprises a mixture of DNA molecules, the method further comprising:
selecting a DNA molecule from the mixture likely to have the correct DNA sequence, and using the selected DNA molecule in the synthesis of the DNA sequence.
- 44. The DNA sequence of claim 43, wherein a DNA molecule is separated from the mixture by cloning.
- 45. The DNA sequence of claim 43, wherein the selection comprises sequencing a sample of DNA molecules from the mixture and selecting a DNA molecule with the desired DNA sequence.
- 46. The DNA sequence of claim 43, wherein the selection comprises expressing a polypeptide from each member of a sample of DNA molecules from the mixture, determining the molecular weight of the polypeptide, and selecting a DNA molecule from which a polypeptide with a predetermined molecular weight is expressed.
- 47. The DNA sequence of claim 46, wherein a start codon and/or a stop codon is incorporated into the DNA molecule from which a polypeptide is expressed.
- 48. The DNA sequence of claim 47, wherein the reading frame of the DNA molecule is adjusted with respect to the start codon and/or stop codon.
- 49. The DNA sequence of claim 46, wherein each member of the sample of DNA molecules is inserted into an expression vector, and wherein the expression vector comprises a stop codon downstream from the inserted DNA molecule.
- 50. The DNA sequence of claim 46, wherein the molecular weight of the polypeptide is determined by electrophoresis.
- 51. The DNA sequence of claim 42, wherein the DNA sequence comprises a regulatory sequence.
- 52. The DNA sequence of claim 42, wherein the DNA sequence comprises an intergenic sequence.
- 53. The DNA sequence of claim 42, wherein the DNA sequence encodes a polypeptide.
- 54. The DNA sequence of claim 53, wherein the polypeptide is a full-length protein.
- 55. The DNA sequence of claim 42, wherein dividing the DNA sequence into small pieces of DNA is performed in a single division.
- 56. The DNA sequence of claim 42, wherein dividing the DNA sequence into small pieces of DNA is performed in a plurality of divisions.
- 57. The DNA sequence of claim 56, wherein the DNA sequence is divided into pieces of DNA of about 1,500 bases long or shorter.
- 58. The DNA sequence of claim 42, wherein the small pieces of DNA are about 60 bases long or shorter.
- 59. The DNA sequence of claim 58, wherein the small pieces of DNA are about 50 bases long or shorter.
- 60. The DNA sequence of claim 42, wherein the overlapping regions comprise from about 6 to about 60 base-pairs.
- 61. The DNA sequence of claim 42, wherein optimizing comprises calculating a melting temperature for the pieces of DNA.
- 62. The DNA sequence of claim 42, wherein optimizing comprises calculating a parameter related to hybridization propensity for the pieces of DNA.
- 63. The DNA sequence of claim 42, wherein the parameter selected from the group consisting of free energy, enthalpy, entropy, and arithmetic or algebraic combinations thereof.
- 64. The DNA sequence of claim 61, wherein the melting temperature of the lowest melting correct hybridization is at least 1° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 65. The DNA sequence of claim 61, wherein the melting temperature of the lowest melting correct hybridization is at least 4° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 66. The DNA sequence of claim 61, wherein the melting temperature of the lowest melting correct hybridization is at least 8° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 67. The DNA sequence of claim 61, wherein the melting temperature of the lowest melting correct hybridization is at least 16° C. higher than the melting temperature of the highest melting incorrect hybridization.
- 68. The DNA sequence of claim 51, wherein optimizing comprises taking advantage of the degeneracy in the regulatory region consensus sequence.
- 69. The DNA sequence of claim 52, wherein optimizing comprises direct base assignment.
- 70. The DNA sequence of claim 52, wherein optimizing comprises adjusting boundary point between adjacent pieces of DNA.
- 71. The DNA sequence of claim 53, wherein optimizing comprises permuting silent codon substitutions.
- 72. The DNA sequence of claim 42, wherein at least one of the optimized small pieces of DNA is synthetic.
- 73. The DNA sequence of claim 42, wherein at least one of the optimized small pieces of DNA is single-stranded.
- 74. The DNA sequence of claim 42, wherein a single-stranded DNA segment has a length of from about zero bases to about 20 bases.
- 75. The DNA sequence of claim 42, wherein the next-larger piece of DNA is produced by cloning the DNA construct.
- 76. The DNA sequence of claim 75, wherein the cloning is selected from the group consisting of exonuclease III cloning, topoisomerase cloning, restriction enzyme cloning, and homologous recombination cloning.
- 77. The DNA sequence of claim 42, wherein the next-larger piece of DNA is produced by ligating the DNA construct.
- 78. The DNA sequence of claim 42, wherein the next-larger piece of DNA is produced by extending the DNA construct by a reaction using DNA polymerase.
- 79. The DNA sequence of claim 78, wherein the DNA polymerase is a proof-reading DNA polymerase.
- 80. The DNA sequence of claim 78, further comprising mixing a DNA polymerase primer with the pieces of DNA derived from the division of the next-larger piece of DNA.
- 81. The DNA sequence of claim 42, further comprising designing a restriction site into an overlapping region.
- 82. The DNA sequence of claim 81, further comprising digesting the restriction site with a site-specific restriction enzyme.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/472,822, filed on May 22, 2003, the disclosure of which is incorporated by reference.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60472822 |
May 2003 |
US |