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1. WO2012040628 - RAPID ASSEMBLY OF MULTIPLE ARBITRARY LENGTH DNA FRAGMENTS

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[ EN ]

CLAIMS

1. A method of assembling a plurality of DNA molecules, comprising:

preparing a double- stranded (ds)DNA molecule containing (i) a first 5' overhang DNA sequence of at least 10 nucleotides on a first strand of the dsDNA molecule and (ii) a second 5' overhang DNA sequence of at least 10 nucleotides on a second strand of the dsDNA molecule, wherein the first overhang and the second overhang do not anneal together, and wherein each overhang anneals to one overhang of a second and a third dsDNA molecule, respectively;

preparing a second dsDNA molecule containing (i) a first 5' overhang DNA sequence of at least 10 nucleotides on a first strand of the dsDNA molecule and (ii) a second 5' overhang DNA sequence of at least 10 nucleotides on a second strand of the dsDNA molecule, wherein the first overhang and the second overhang do not anneal together, and wherein each overhang anneals to one overhang of a third and a fourth dsDNA molecule, respectively; and

combining the first dsDNA molecule and the second dsDNA molecule under conditions that permit DNA annealing of the 5' overhang sequences, thereby assembling the plurality of dsDNA molecules.

2. The method of claim 1, wherein the first and second dsDNA molecules are combined with at least one additional dsDNA molecule.

3. The method of claim 1 or claim 2, wherein the first and second 5' overhangs are at least 20 nucleotides in length.

4. The method of any one of claims 1-3, wherein the first and second 5' overhangs are at least 30 nucleotides in length.

5. The method of any one of claims 1-4, wherein the first and second 5' overhangs are at least 40 nucleotides in length.

6. The method of any one of claims 1-5, wherein the first and second 5' overhangs are at least 50 nucleotides in length.

7. The method of any one of claims 1-6, further comprising

preparing a double stranded linear nucleic acid vector containing (i) a first 5' overhang DNA sequence of at least 10 nucleotides on a first strand of the vector and (ii) a second 5' overhang DNA sequence of at least 10 nucleotides on a second strand of the vector, wherein the first overhang and the second overhang do not anneal together, and wherein each overhang anneals to one overhang of two different dsDNA molecules; and

combining the assembled dsDNA molecules of any one of claims 1-6 with the vector under conditions that permit DNA annealing, wherein the assembled dsDNA molecules comprise overhangs that anneal to the overhangs of the vector, thereby assembling the vector and the assembled dsDNA molecules.

8. The method of any one of claims 1-7, further comprising probing one or more of the overhang sequences of assembled dsDNA molecule to select correctly assembled dsDNA molecules.

9. The method of any one of claims 1-8, further comprising probing the overhang sequences of assembled dsDNA molecule to confirm the structure of the assembled dsDNA molecule.

10. A method of preparing a dsDNA molecule, comprising:

providing a circular DNA vector comprising a vector backbone VB and a

polynucleotide P-X-S, wherein P is a prefix nucleic acid sequence and S is a suffix nucleic acid sequence, each comprising one or more enzyme recognition sites, and X is a DNA part of interest;

linearizing the circular DNA vector with one or more enzyme that cleaves one or more enzyme recognition sites in the suffix to produce a linear double-stranded (ds) DNA having a 5' overhang at each end of the dsDNA;

ligating to the 5' overhang of the suffix a first oligonucleotide having a length of at least 20 nucleotides;

removing the vector backbone from the linear dsDNA with one or more enzyme that cleaves one or more enzyme recognition sites in the prefix, producing a 5' overhang; and ligating to the 5' overhang of the prefix a second oligonucleotide having a length of at least 20 nucleotides, thereby preparing a dsDNA molecule.

11. The method of claim 10, wherein the DNA element X is selected from the group consisting of: a terminator sequence, a ribosome binding site sequence, a protein coding sequence, a reporter sequence, a signaling sequence, a primer sequence, and a regulatory sequence.

12. The method of claim 10 or claim 11, wherein the enzymes are selected from the group consisting of EcoRI, XbaI, PstI, and SpeI.

13. The method of claim 10 or claim 11, wherein the enzymes are selected from I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI- PƒuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, l-SceII, l-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp68031, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, and I-Vdil41I.

14. The method of any one of claims 10-13, further comprising probing the prefix and/or the suffix of the newly assembled DNA molecule to select correctly assembled dsDNA molecules.

15. The method of any one of claims 10-14, further comprising probing the prefix, then probing the suffix of the newly assembled DNA molecule to confirm the structure of the assembled dsDNA molecule.

16. The method of any one of claims 1-15, wherein the method excludes polymerase chain reaction (PCR) purification.

17. The method of any one of claims 1-16, wherein the method excludes antibiotic selection.

18. The method of any one of claims 1-17, wherein the method is automated.

19. A DNA molecule, comprising

a double-stranded DNA;

a first 5' overhang of at least 10 nucleotides in length located at one end of the DNA; and

a second 5' overhang of at least 10 nucleotides in length located at the other end of the DNA.

20. The DNA molecule of claim 19, wherein the double-stranded DNA is a standard DNA part.

21. A DNA molecule, comprising a first biotinylated oligonucleotide of at least 10 nucleotides in length, wherein the first oligonucleotide is not phosphorylated and is attached to a bead.

22. The DNA molecule of claim 21, wherein the bead is a streptavidin-coated bead.

23. The DNA molecule of claim 21 or claim 22, further comprising a second

oligonucleotide of at least 10 nucleotides in length annealed to the first oligonucleotide such that there is a 5' overhang opposite the bead.

24. A double-stranded DNA molecule comprising a nucleic acid of interest flanked by a prefix sequence and suffix sequence, each of the prefix sequence and the suffix sequence comprising one or more homing enzyme restriction sites.

25. The DNA molecule of claim 24, further comprising an origin of replication.

26. The DNA molecule of claim 24 or claim 25, further comprising an antibiotic resistance selection marker.

27. The DNA molecule of any one of claims 24-26, wherein the double stranded molecule is attached to a bead or a DNA molecule comprising a bead.

28. A collection of two or more of the DNA molecules of any one of claims 21-27.

29. A kit comprising one or more containers containing two or more of the DNA molecules of any one of claims 21-27.