Methods for the enzymatic assembly of polynucleotides and identification of polynucleotides having desired characteristics
AssigneeHercules Incorporated
InventorsSimon Delagrave, Barry Marrs
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Abstract
The present invention provides methods of preparing large polynucleotides of arbitrary sequence and in a manner that will readily lend itself to automation. The present invention provides methods of preparing a polynucleotide having at least 200 nucleotides in either a 5′ to 3′ or 3′ to 5′ direction by ligating a plurality of oligonucleotides, the assembly of which, represents the nucleotide sequence of the desired polynucleotide. The present invention also provides libraries of polynucleotides and screening of libraries for polynucleotide members having desired properties.
Claims26
A method of preparing a polynucleotide from a plurality of oligonucleotides, said method comprising:
- a) blocking the 3′ terminus of a first oligonucleotide with a blocking group to form a blocked oligonucleotide, wherein said first oligonucleotide comprises the 3′ terminus of said polynucleotide;
- b) coupling the 3′ terminus of a further oligonucleotide from said plurality of oligonucleotides to the 5′ terminus of said blocked oligonucleotide to form a coupled oligonucleotide;
- wherein said coupling is carried out in the presence of an RNA ligase or a ribozyme;
- c) phosphorylating the 5′ terminus of said further oligonucleotide;
- d) amplifying said coupled oligonucleotide to form an amplified oligonucleotide substantially free of blocking group;
- and
- e) repeating steps a) to d) with said amplified oligonucleotide until said polynucleotide is prepared.
A method of preparing a polynucleotide from a plurality of oligonucleotides, said method comprising:
- a) blocking the 3′ terminus of each of said oligonucleotides, except for an unblocked oligonucleotide comprising the 5′ terminus of said polynucleotide, with a blocking group to form a plurality of blocked oligonucleotides;
- b) phosphorylating the 5′ terminus of each of said blocked oligonucleotides;
- c) coupling the 3′ terminus of said unblocked oligonucleotide with the 5′ terminus of one of said blocked oligonucleotides, wherein said coupling is carried out in the presence of an RNA ligase or a ribozyme;
- d) amplifying said coupled oligonucleotides to form an amplified oligonucleotide substantially free of blocking groups;
- and
- e) repeating steps c) and d) with said amplified oligonucleotide until said polynucleotide is prepared.
Dependencies are inferred from the claim text.