Claims
- 1. A method of cloning a target DNA into a vector, the method comprising:
providing a first megaprimer; providing a second megaprimer; providing one or more nucleic acid that comprises or encodes the target DNA, the one or more nucleic acid comprising at least one region of complementarity to or identity with the first megaprimer and at least one region of complementarity to or identity with the second megaprimer; extending the megaprimers; and, intramolecularly ligating the extended product to form a functional vector.
- 2. The method of claim 1, wherein the one or more nucleic acid consists of one nucleic acid that at a first end comprises at least one region of complementarity to or identity with the first megaprimer and at a second end comprises at least one region of complementarity to or identity with the second megaprimer.
- 3. The method of claim 1, wherein the one or more nucleic acid comprises at least two nucleic acids, wherein an end of at least one of the at least two nucleic acids comprises at least one region of complementarity to or identity with the first megaprimer and an end of at least one of the at least two nucleic acids comprises at least one region of complementarity to or identity with the second megaprimer.
- 4. The method of claim 1, wherein the functional vector is double-stranded.
- 5. The method of claim 1, wherein the ligation is performed in vitro.
- 6. The method of claim 1, wherein the first and second megaprimers each comprise a nonfunctional marker or a fragment thereof.
- 7. The method of claim 6, wherein the intramolecular ligation forms a functional marker.
- 8. The method of claim 7, wherein the marker comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 9. The method of claim 7, comprising transforming the vector into cells and selecting or screening the cells for expression of the marker.
- 10. The method of claim 1, wherein either the first or the second megaprimer comprises a nonfunctional marker or a fragment thereof and the one or more nucleic acid comprises a replacement sequence comprising a portion of the marker or its reverse complement, wherein integration of the replacement sequence with the nonfunctional marker results in a functional marker.
- 11. The method of claim 10, wherein the nonfunctional marker comprises a mutation of a functional marker comprising at least one mutation selected from the group consisting of: a deletion, an insertion, and a point mutation.
- 12. The method of claim 10, wherein the functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 13. The method of claim 10, wherein the target DNA comprises an open reading frame located 5′ of and in frame with the replacement sequence.
- 14. The method of claim 10, comprising transforming the extended product into cells and selecting or screening the cells for expression of the marker resulting from integration.
- 15. The method of claim 1, wherein the one or more nucleic acid is a single-stranded DNA comprising or encoding the target DNA, the single-stranded DNA comprising at least one region identical to a region of the first megaprimer 5′ of the target DNA and at least one region complementary to the second megaprimer 3′ of the target DNA.
- 16. The method of claim 15, wherein the single-stranded DNA is a chemically synthesized oligonucleotide.
- 17. The method of claim 15, wherein extending the megaprimers comprises annealing the single-stranded DNA to the second megaprimer, extending the second megaprimer, annealing the extended second megaprimer to the first megaprimer, and extending the first megaprimer and extended second megaprimer.
- 18. The method of claim 17, comprising denaturing the double-stranded product formed by extending the second megaprimer prior to annealing the extended second megaprimer to the first megaprimer.
- 19. The method of claim 1, wherein the one or more nucleic acid is double-stranded DNA.
- 20. The method of claim 1, comprising digesting with at least one restriction enzyme prior to the intramolecular ligation step.
- 21. A method of cloning a target DNA into a vector, the method comprising:
providing a first vector or vector template comprising a nonfunctional marker or fragment thereof; h providing one or more nucleic acid comprising or encoding the target DNA, the one or more nucleic acid comprising at least one region complementary to a strand of the first vector or vector template and a replacement sequence comprising a portion of the marker or its reverse complement, wherein integration of the replacement sequence with the nonfunctional marker results in a functional marker; annealing the one or more nucleic acid to the first vector or vector template; extending the one or more nucleic acid; denaturing the resulting extended product; providing an extension primer capable of annealing to both 5′ and 3′ ends of the extended product; annealing the extension primer to the extended product; extending the extension primer; and intramolecularly ligating the doubly-extended product to form a vector comprising a functional marker.
- 22. The method of claim 21, wherein the first vector or vector template is a double-stranded vector, and wherein the double-stranded vector is denatured prior to annealing the one or more nucleic acid to the double-stranded vector.
- 23. The method of claim 21, wherein the one or more nucleic acid consists of one nucleic acid.
- 24. The method of claim 21, wherein the one or more nucleic acid comprises at least two nucleic acids.
- 25. The method of claim 21, wherein the nonfunctional marker comprises a mutation of a functional marker comprising at least one mutation selected from the group consisting of: a deletion, an insertion, and a point mutation.
- 26. The method of claim 21, wherein the functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 27. The method of claim 21, wherein the DNA polymerase used to extend the one or more nucleic acid or the extension primer lacks strand displacement or 5′ to 3′ exonuclease activity.
- 28. The method of claim 21, wherein the ligation is performed in vitro.
- 29. The method of claim 28, comprising transforming the ligated doubly-extended product into cells and selecting or screening the cells for expression of the marker.
- 30. The method of claim 21, wherein the one or more nucleic acid is a chemically synthesized oligonucleotide that is at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides in length.
- 31. The method of claim 30, wherein the replacement sequence is proximal to the 5′ end of the oligonucleotide.
- 32. The method of claim 31, wherein the 5′ end of the oligonucleotide anneals before the 3′ end.
- 33. The method of claim 21, wherein the first vector or vector template comprises a second nonfunctional marker or fragment thereof and the one or more nucleic acid comprises a second replacement sequence comprising a portion of the second marker or its reverse complement, wherein integration of the second replacement sequence with the second nonfunctional marker results in a second functional marker.
- 34. The method of claim 33, wherein the target DNA comprises an open reading frame located 5′ of and in frame with the second replacement sequence.
- 35. The method of claim 33, wherein the second functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein
- 36. The method of claim 33, comprising transforming the doubly-extended product into cells and selecting or screening the cells for expression of the second marker resulting from integration of the second replacement sequence with the second non-functional marker.
- 37. The method of claim 21, comprising denaturing the one or more nucleic acid prior to annealing the one or more nucleic acid to the first vector or vector template.
- 38. The method of claim 21, wherein the first vector or vector template comprises a functional selectable marker.
- 39. A method of cloning a target DNA into a vector, the method comprising:
providing a linear first vector or vector template comprising a nonfunctional marker or fragment thereof; providing one or more nucleic acid comprising or encoding the target DNA, the one or more nucleic acid comprising at least one region complementary to a strand of the first vector or vector template and a replacement sequence comprising a portion of the marker or its reverse complement, wherein integration of the replacement sequence with the nonfunctional marker results in a functional marker; annealing the one or more nucleic acid to the first vector or vector template; extending the one or more nucleic acid; denaturing the resulting extended product; providing a primer comprising the reverse complement of the 3′ end of the extended product; annealing the primer to the extended product; extending the primer; and, intramolecularly ligating the doubly-extended product to form a functional vector comprising a functional marker.
- 40. The method of claim 39, wherein the linear first vector or vector template is a linear double-stranded vector, and wherein the linear double-stranded vector is denatured prior to annealing the one or more nucleic acid.
- 41. The method of claim 40, wherein the linear double-stranded vector is produced by digestion with at least one restriction enzyme that cleaves a site located within the nonfunctional marker.
- 42. The method of claim 39, wherein the one or more nucleic acid consists of one nucleic acid
- 43. The method of claim 39, wherein the one or more nucleic acid comprises at least two nucleic acids.
- 44. The method of claim 39, wherein the nonfunctional marker comprises a mutation of a functional marker comprising at least one mutation selected from the group consisting of: a deletion, an insertion, and a point mutation.
- 45. The method of claim 39, wherein the functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 46. The method of claim 39, wherein the DNA polymerase used to extend the one or more nucleic acid or the primer lacks strand displacement or 5 to 3′ exonuclease activity.
- 47. The method of claim 39, wherein the ligation is performed in vitro.
- 48. The method of claim 47, comprising transforming the ligated doubly-extended product into cells and selecting or screening the cells for expression of the marker.
- 49. The method of claim 39, wherein the one or more nucleic acid is a chemically synthesized oligonucleotide that is at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides in length.
- 50. The method of claim 49, wherein the replacement sequence is proximal to the 5′ end of the oligonucleotide.
- 51. The method of claim 39, wherein the linear first vector or vector template comprises a second nonfunctional marker or fragment thereof and the one or more nucleic acid comprises a second replacement sequence comprising a portion of the second marker or its reverse complement, wherein integration of the second replacement sequence with the second nonfunctional marker results in a second functional marker.
- 52. The method of claim 51, wherein the target DNA comprises an open reading frame located 5′ of and in frame with the second replacement sequence.
- 53. The method of claim 51, wherein the second functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 54. The method of claim 51, comprising transforming the doubly-extended product into cells and selecting or screening the cells for expression of the second marker.
- 55. The method of claim 39, comprising denaturing the one or more nucleic acid prior to annealing the one or more nucleic acid to the first vector or vector template.
- 56. The method of claim 39, comprising digesting the doubly-extended product with at least one restriction enzyme prior to the intramolecular ligation.
- 57. The method of claim 39, wherein the first vector or vector template comprises a functional selectable marker.
- 58. A method of cloning a target DNA into a vector, the method comprising:
providing a first vector or vector template comprising a nonfunctional marker or fragment thereof; providing one or more nucleic acid comprising or encoding the target DNA, the one or more nucleic acid comprising at least one region complementary to a strand of the first vector or vector template and a replacement sequence comprising a portion of the marker or its reverse complement, wherein integration of the replacement sequence with the nonfunctional marker results in a functional marker; annealing the one or more nucleic acid to the first vector or vector template; extending the one or more nucleic acid; and, intramolecularly ligating the extended product to form a vector comprising a functional marker.
- 59. The method of claim 58, wherein the first vector or vector template is a double-stranded vector, and wherein the double-stranded vector is denatured prior to annealing the one or more nucleic acid to the double-stranded vector.
- 60. The method of claim 58, wherein the one or more nucleic acid consists of one nucleic acid.
- 61. The method of claim 58, wherein the one or more nucleic acid comprises at least two nucleic acids.
- 62. The method of claim 58, wherein the nonfunctional marker comprises a mutation of a functional marker comprising at least one mutation selected from the group consisting of: a deletion, an insertion, and a point mutation.
- 63. The method of claim 58, wherein the functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 64. The method of claim 58, wherein the DNA polymerase used to extend the one or more nucleic acid lacks strand displacement or 5′ to 3′ exonuclease activity.
- 65. The method of claim 58, wherein the ligation is performed in vitro.
- 66. The method of claim 65, comprising transforming the ligated extended product into cells capable of tolerating heteroduplexes and selecting or screening the cells for expression of the marker.
- 67. The method of claim 58, wherein the one or more nucleic acid is a chemically synthesized oligonucleotide that is at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides in length.
- 68. The method of claim 67, wherein the replacement sequence is proximal to the 5′ end of the oligonucleotide.
- 69. The method of claim 58, wherein the first vector or vector template comprises a second nonfunctional marker or fragment thereof and the one or more nucleic acid comprises a second replacement sequence comprising a portion of the second marker or its reverse complement, wherein integration of the second replacement sequence with the second nonfunctional marker results in a second functional marker.
- 70. The method of claim 69, wherein the target DNA comprises an open reading frame located 5′ of and in frame with the second replacement sequence.
- 71. The method of claim 69, wherein the second functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 72. The method of claim 69, comprising transforming the extended product into cells capable of tolerating heteroduplexes and selecting or screening the cells for expression of the second marker.
- 73. The method of claim 58, comprising denaturing the one or more nucleic acid prior to annealing the one or more nucleic acid to the first vector or vector template.
- 74. The method of claim 58, wherein the first vector or vector template comprises a functional selectable marker.
- 75. 75. A method of cloning a target DNA into a vector, the method comprising:
providing a first vector or vector template; providing a first chemically synthesized oligonucleotide that is at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides in length that comprises or encodes the target DNA, the first oligonucleotide comprising a first restriction site 5′ of the target and a region of sequence that is complementary to a first strand of the first vector or vector template 3′ of the target; providing a second oligonucleotide primer with a second restriction site 5′ of a region of sequence complementary to a second strand of the first vector or vector template; performing at least one cycle of PCR amplification to extend the provided oligonucleotides; digesting the double-stranded product with a first restriction enzyme that cleaves the first restriction site; digesting the double-stranded product with a second restriction enzyme that cleaves the second restriction site; and ligating the digested product.
- 76. The method of claim 75, wherein the first vector or vector template is a double-stranded vector.
- 77. The method of claim 75, wherein the first and second restriction sites are identical and the first and second restriction enzymes are identical.
- 78. The method of claim 75, wherein the first vector or vector template comprises a nonfunctional marker or fragment thereof and the first oligonucleotide comprises a replacement sequence comprising a portion of the marker or its reverse complement, wherein integration of the replacement sequence with the nonfunctional marker results in a functional marker, the replacement sequence located proximal to the 3′ end of the first oligonucleotide.
- 79. The method of claim 78, wherein the nonfunctional marker comprises a mutation of a functional marker comprising at least one mutation selected from the group consisting of: a deletion, an insertion, and a point mutation.
- 80. The method of claim 78, wherein the functional marker resulting from integration comprises one or more of: a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, a gene conferring resistance to neomycin, an optically detectable marker, a marker nucleic acid that encodes a green fluorescent protein, or a marker nucleic acid that encodes a beta galactosidase protein.
- 81. The method of claim 78, wherein the target DNA comprises an open reading frame located 5′ of and in frame with the replacement sequence.
- 82. The method of claim 78, comprising transforming the ligated product into cells and selecting or screening the cells for expression of the marker.
- 83. The method of claim 75, wherein the ligation is performed in vitro.
- 84. The method of claim 75, further comprising providing a third oligonucleotide primer comprising a region of sequence identical to the 5′ region of the first oligonucleotide.
- 85. The method of claim 75, comprising digesting the double-stranded product with an enzyme that cleaves the provided first vector or vector template but not the product of the PCR amplification.
- 86. The method of claim 85, wherein the enzyme is Dpn I.
- 87. The method of claim 75, wherein the first vector or vector template comprises a functional selectable marker.
- 88. A method of making a double-stranded DNA, the method comprising:
chemically synthesizing a plurality of oligonucleotides that are each at least 100 nucleotides in length and that collectively comprise a plurality of subsequences of the double stranded DNA; assembling the plurality of oligonucleotides to form a plurality of genomers; assembling the genomers to form the double-stranded DNA; and determining at least one property of the double-stranded DNA.
- 89. The method of claim 88, wherein each of the plurality of oligonucleotides is at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides in length.
- 90. The method of claim 88, wherein the genomers are double-stranded.
- 91. The method of claim 88, wherein the at least one property of the double-stranded DNA is determined by one or more of: sequencing the DNA, restriction enzyme digestion of the DNA, or screening for the expression of a marker fused to the DNA.
- 92. The method of claim 88, comprising purifying the plurality of oligonucleotides.
- 93. The method of claim 92, wherein the oligonucleotides are purified by enzymatic cleavage or by photocleavage.
- 94. The method of claim 88, comprising determining at least one property of one or more of the genomers prior to assembling the genomers to form the double-stranded DNA.
- 95. The method of claim 94, wherein the at least one property of the genomer is determined by one or more of: sequencing the genomer, restriction enzyme digestion of the genomer, or screening for expression of a marker fused to the genomer.
- 96. A method for purifying a target oligonucleotide, comprising:
providing a tagged target oligonucleotide comprising a target oligonucleotide sequence and a tag sequence 5′ of the target sequence; providing a bait oligonucleotide comprising a region complementary to the tag; annealing the tagged target oligonucleotide and bait oligonucleotide; and digesting the annealed oligonucleotides with a nicking endonuclease that cleaves the tagged target oligonucleotide at a junction between the 3′ end of the tag sequence and the 5′ end of the target sequence.
- 97. The method of claim 96, wherein the nicking endonuclease cleaves at a site that is 3′ of its recognition sequence.
- 98. The method of claim 96, wherein the nicking endonuclease is N.BstNBI or N.AlwI.
- 99. The method of claim 96, wherein the bait oligonucleotide comprises a means for attaching the bait oligonucleotide to a solid support, and wherein the bait oligonucleotide is attached to the solid support before or after annealing the tagged target oligonucleotide and bait oligonucleotide.
- 100. A composition comprising: a pair of megaprimers, the pair comprising a first megaprimer and a second megaprimer, wherein each megaprimer is a single-stranded DNA molecule that comprises a distinct portion of a vector backbone and a distinct portion of an essential marker.
- 101. The composition of claim 100, wherein the essential marker comprises one or more of: a sequence element required for replication of a plasmid, an origin of replication, a selectable marker, a gene that confers cellular resistance to an antibiotic, a gene conferring resistance to ampicillin, a gene conferring resistance to tetracycline, a gene conferring resistance to kanamycin, or a gene conferring resistance to neomycin.
- 102. The composition of claim 100, wherein a first portion of the essential marker is proximal to the 5′ end of the first megaprimer and a second portion of the essential marker is proximal to the 5′ end of the second megaprimer.
- 103. The composition of claim 100, wherein the vector backbone comprises one or more of: an origin of replication, a selectable marker, a nonfunctional marker, an inducible promoter, or a multiple cloning site.
- 104. The composition of claim 100, further comprising one or more chemically synthesized oligonucleotide that comprises or encodes a target DNA.
- 105. A composition comprising:
a vector comprising at least one nonfunctional marker or fragment thereof, and one or more chemically synthesized oligonucleotide, wherein the oligonucleotide is at least 100, at least 150, at least 200, at least 250, or at least 300 nucleotides in length, the one or more oligonucleotide comprising at least one region complementary to at least one region of the vector and a replacement sequence, the replacement sequence comprising a portion of the marker or its reverse complement, wherein integration of the replacement sequence with the nonfunctional marker results in a functional marker.
- 106. A set of synthetic oligonucleotides wherein each synthetic oligonucleotide is at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides in length, wherein the oligonucleotides collectively comprise a genomer, gene, or other full-length DNA of interest.
- 107. The set of synthetic oligonucleotides of claim 106, comprising at least about 2, 5, 10, 20, 48, 96, 384, or 1536 members.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional utility patent application claiming priority to and benefit of the following provisional patent applications: U.S. S No. 60/296,162 filed Jun. 5, 2001, entitled “Methods for the Error Free Synthesis of DNA Molecules” by Beckman et al.; U.S. S No. 60/327,351, filed Oct. 4, 2001, entitled “Method for Cloning Long Oligonucleotides by Oligomer Priming on a Vector Template” by Mancebo et al.; and, U.S. S No. 60/296,038, filed Jun. 5, 2001, entitled “Methods for Very Low Background Cloning of DNA” by Mancebo et al. The present application claims priority to and benefit of each of these prior applications, each of which is incorporated by reference. The present application is also related to U.S. S No. 60/273,812, filed Mar. 6, 2001, entitled “A method for Purifying Full-length DNA Oligonucleotides Using Site-Specific Endonucleases,” by Mancebo et al., which is incorporated herein by reference.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60296162 |
Jun 2001 |
US |
|
60296038 |
Jun 2001 |
US |
|
60327351 |
Oct 2001 |
US |