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1. (WO2018111914) PROCÉDÉS D’ASSEMBLAGE DE MOLÉCULES D’ADN
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

Claims

1. A method of assembling a DNA molecule having a desired sequence comprising: a) contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides,

wherein each oligonucleotide of a pair comprises a portion of the desired sequence, and the oligonucleotides of a pair comprise an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair;

wherein when the plurality of pairs of oligonucleotides are arranged in order, adjacent to each other and according to their internal sequences they comprise at least a portion of the desired sequence;

wherein the oligonucleotides comprise a 3' or a 5' primer binding sequence;

wherein the desired sequence has a 3' end and a 5' end, and the oligonucleotide pairs that comprise the 3' and 5' ends of the desired sequence further comprise a universal 3' flanking sequence and a universal 5' flanking sequence, respectively; and

b) performing a first amplification reaction on the plurality of pairs of oligonucleotides;

c) removing the 3' and 5' primer binding sequences from the plurality of pairs of oligonucleotides; and

d) subjecting the plurality of pairs of oligonucleotides to an assembly reaction to thereby assemble the dsDNA molecule having the desired sequence.

2. The method of claim 1 wherein the first amplification reaction further comprises primers that bind to the 3' and 5' primer binding sequences.

3. The method of claim 1 wherein the universal 3' flanking sequence and the universal 5' flanking sequence are comprised to the inside of the 3' and 5' flanking sequences, respectively.

4. The method of claim 1 further comprising a step of removing at least a portion of the universal 3' flanking sequence and the universal 5' flanking sequence.

5. The method of claim 1 wherein the first amplification reaction comprises a denaturation phase, an annealing phase, and an extension phase.

6. The method of claim 5 wherein the amplification reaction is the polymerase chain reaction, or a variant of PCR.

7. The method of claim 1 further comprising a second, and optionally additional, amplification reactions.

8. The method of claim 1 wherein the 5' and 3' primer binding sequences on an oligonucleotide pair are not complementary to each other.

9. The method of claim 1 wherein the method assembles a plurality of DNA molecules of desired sequences.

10. The method of claim 9 wherein the plurality of DNA molecules comprises a plurality of distinct genes.

11. The method of claim 1 wherein the pairs of oligonucleotides are comprised on a solid support.

12. The method of claim 11 wherein the solid support is a nucleic acid array.

13. The method of claim 12 wherein at least 15 pairs of oligonucleotides are comprised on the array.

14. The method of claim 1 wherein the oligonucleotides comprise from 60 to 100 nucleotides and the primer binding sequences comprise from 8 to 30 nucleotides.

15. The method of claim 14 wherein the dsDNA molecule assembled is a nucleic acid construct selected from the group consisting of: a plasmid, an artificial chromosome, and a functional gene.

16. The method of claim 6 wherein a first pair of oligonucleotides comprises a first set of 3' or 5' primer binding sequences, and a second pair of oligonucleotides comprises a second set of 3' or 5' primer binding sequences.

17. The method of claim 2 wherein the 3' and 5' primer binding sequences do not comprise a restriction site for a restriction enzyme.

18. The method of claim 2 wherein the 3' and 5' primer binding sequences are removed by the action of one or more enzymes that specifically cleave the primer binding sequences, and wherein the one or more enzymes do not comprise a restriction enzyme.

19. The method of claim 18 wherein the one or more enzymes comprise uracil DNA glycosylase, endonuclease VIII, and exonuclease T.

20. The method of claim 1 wherein the desired DNA molecule comprises a predetermined sequence.

21. The method of claim 2 wherein a plurality of oligonucleotide pairs is comprised in a single container and forms at least two couplets, and the at least two couplets comprise distinct sets of 3' or 5' primer binding sequences, and the primers comprise two sets of primers that bind specifically to the at least two couplets.

22. The method of claim 1 further comprising a step of forming the plurality of pairs of oligonucleotides into a plurality of couplets.

23. The method of claim 1 performed in a single container.

24. The method of claim 1 which is an automated method.

25. The method of claim 1 wherein the oligonucleotides comprised in the pairs of oligonucleotides are from about 50 to about 200 nucleotides in length.

26. The method of claim 1 wherein the dsDNA molecule assembled in the method is greater than 5 Mbp in size.

27. The method of claim 1 wherein the nucleic acid molecule of desired sequence is a scarless DNA molecule.

28. A composition comprising

a DNA polymerase, dNTPs, and a plurality of oligonucleotides formed into couplets, wherein each couplet comprises an internal sequence that comprises a portion of the desired nucleic acid sequence;

wherein when the plurality of couplets is arranged in order, adjacent to each other and according to their internal sequences they comprise at least a portion of a desired nucleic acid sequence, and each couplet further comprises a 3' or a 5' primer binding sequence, and each couplet contains a sequence that overlaps and is complementary to a portion of a sequence from an adjacent couplet; and

wherein the desired nucleic acid sequence has a 3' end and a 5' end, and the couplets that comprise the 3' and 5' ends of the desired sequence further comprise a universal 3' flanking sequence and a universal 5' flanking sequence, respectively.

29. The composition of claim 28 comprised in a single container and further comprising an effective amount of a preservative.

30. The composition of claim 30 wherein the couplets comprise at least 50% of the oligonucleotides in the mixture, and the couplets overlap at least 33% of their sequences.