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US 9,790,487 B2Granted
Rapid assembly of multiple arbitrary length DNA fragments
AssigneeMassachusetts Institute of Technology
InventorsJonathan William Babb, Shuo Cory Li, Thomas Knight, Ron Weiss
Life of this patent
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Abstract
Aspects herein relate to composition, and related methods, for isolating and assembling DNA molecules without intermediate cloning steps.
Claims26
A method of preparing a double-stranded (ds)DNA molecule, comprising:
- (a) providing a first and a second circular DNA vector, each comprising a vector backbone VB and a polynucleotide P-X-S, wherein P is a prefix nucleic acid sequence comprising an enzyme recognition site and S is a suffix nucleic acid sequence comprising an enzyme recognition site, and X is a DNA part of interest;
- (b) linearizing the first and second circular DNA vectors with an enzyme that cleaves the enzyme recognition site in the suffix of the first circular DNA vector and cleaves the enzyme recognition site in the prefix of the second circular DNA vector to produce a first and a second linear double-stranded (ds) DNA, each having a 5′ overhang at each end of the linear dsDNA;
- (c) ligating the 5′ overhang of the suffix of the first linear dsDNA to the 5′ overhang of the prefix of the second linear dsDNA;
- (d) removing the vector backbones from the linear dsDNAs with one or more enzyme(s) that cleave(s) the enzyme recognition site in the prefix of the first linear dsDNA and in enzyme recognition site in the suffix of the second linear dsDNA to produce a single linear dsDNA comprising a prefix having a 5′ overhang and a suffix having a 5′ overhang;
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- (e) ligating to the 5′ overhang of the prefix of the single linear dsDNA of (d) a first oligonucleotide having a length of at least 20 nucleotides and ligating to the 5′ overhang of the suffix of the single linear dsDNA of (d) a second oligonucleotide having a length of at least 20 nucleotides, thereby preparing a dsDNA molecule having a 5′ overhang of at least 20 nucleotides at each end of the dsDNA.
3 more elements
The method of claim12, wherein the bead is a magnetic bead.
A method of preparing a double-stranded (ds)DNA molecule, comprising:
- (a) providing a first, second, and at least one other circular DNA vector, each comprising a vector backbone VB and a polynucleotide P-X-S, wherein P is a prefix nucleic acid sequence comprising an enzyme recognition site and S is a suffix nucleic acid sequence comprising an enzyme recognition site, and X is a DNA part of interest;
- (b) linearizing the first, second, and at least one other circular DNA vectors with (i) a first enzyme that cleaves the enzyme recognition site in the suffix of the first circular DNA vector and cleaves the enzyme recognition site in the prefix of the second circular DNA vector and (ii) a second enzyme, having a different recognition site from the first enzyme, that cleaves the enzyme recognition site in the suffix of the second circular DNA vector and cleaves the enzyme recognition site in the prefix of the at least one other circular DNA vector to produce a first, a second, and at least one other linear double-stranded (ds) DNA, each having a 5′ overhang at each end of the linear dsDNA;
- (c) ligating the 5′ overhang of the suffix of the first linear dsDNA to the 5′ overhang of the prefix of the second linear dsDNA, ligating the 5′ overhang of the suffix of the second linear dsDNA to the 5′ overhang of the prefix of the at least one other linear dsDNA, to form a single linear dsDNA having a prefix and a suffix;
- (d) removing the vector backbones from the single linear dsDNA with one or more enzyme(s) that cleave(s) the enzyme recognition site in the prefix of the single linear dsDNA and the enzyme recognition site in the suffix of the single linear dsDNA to produce a single linear dsDNA comprising a prefix having a 5′ overhang and a suffix having a 5′ overhang;
- and
- (e) ligating to the 5′ overhang of the prefix of the single linear dsDNA of (d) a first oligonucleotide having a length of at least 20 nucleotides and ligating to the 5′ overhang of the suffix of the single linear dsDNA of (d) a second oligonucleotide having a length of at least 20 nucleotides, thereby preparing a dsDNA molecule that comprises the X of the first, the second, the at least one other circular DNA vector and a 5′ overhang of at least 20 nucleotides at each end of the dsDNA.
3 more elements
Dependencies are inferred from the claim text.