Rapid assembly of multiple arbitrary length DNA fragments

Patent metadata

Field	Value
Publication number	US-9487808-B2
Application number	US-201113825684-A
Country	US
Kind code	B2
Filing date	Sep 23, 2011
Priority date	Sep 24, 2010
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

Aspects herein relate to composition, and related methods, for isolating and assembling DNA molecules without intermediate cloning steps.

First claim

What is claimed is:

1. A method of preparing a double-stranded (ds)DNA 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.

2. The method of claim 1 , wherein the DNA part 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.

3. The method of claim 1 , further comprising probing the prefix and/or the suffix of the newly prepared dsDNA molecule to select a correctly prepared dsDNA molecule.

4. The method of claim 1 , further comprising probing the prefix, then probing the suffix of the newly prepared dsDNA molecule to confirm the structure of the prepared dsDNA molecule.

5. The method of claim 1 , wherein the circular DNA vector comprises an origin of replication and/or an antibiotic resistance selection marker.

6. The method of claim 1 , wherein the one or more enzyme recognition site(s) is a Type II enzyme recognition site.

7. The method of claim 6 , wherein the Type II enzyme recognition site is a EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3, APovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SpeI, SphI, StuI or XbaI recognition site.

8. The method of claim 1 , wherein the one or more enzyme recognition site(s) is a homing enzyme recognition site.

9. The method of claim 8 , wherein the homing enzyme recognition site is a 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-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, I-Vdi141I, PI-PspI or PI-IsceI recognition site.

10. A method of assembling a plurality of DNA molecules, comprising:
preparing a first 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, wherein the first 5′ overhang DNA sequence of at least 10 nucleotides on the first strand of the first and/or second dsDNA molecule is biotinylated, and wherein the first strand of the first and/or second dsDNA molecule is not phosphorylated and is attached to a solid phase.

11. The method of claim 10 , wherein the solid phase is a bead.

12. The method of claim 11 , wherein the bead is a magnetic bead.

13. The method of claim 10 , wherein the solid phase is a glass surface.

14. A method of assembling a plurality of DNA molecules, comprising:
preparing a first 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, wherein at least one dsDNA molecule of the plurality is attached to a solid phase or a DNA molecule comprising a solid phase.

15. The method of claim 14 , wherein the solid phase is a bead.

16. The method of claim 15 , wherein the bead is a magnetic bead.

17. The method of claim 14 , wherein the solid phase is a glass surface.

Assignees

• Babb Jonathan William
• Li Shuo Cory
• Knight Thomas
• Weiss Ron
• Massachusetts Inst Technology

Inventors

• Babb Jonathan William
• Li Shuo Cory
• Knight Thomas
• Weiss Ron

Classifications

• C12N15/1027 Primary

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title