Solid phase enzymatic assembly of polynucleotides
First Claim
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1. A method of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising the steps:
- a) providing a solid support;
b) providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of said oligonucleotides comprises the nucleotide sequence of said polynucleotide;
c) contacting said solid support with the 3′
terminus of a first oligonucleotide from said plurality of oligonucleotides to form a tethered oligonucleotide;
d) ligating the 3′
terminus of another oligonucleotide from said plurality of oligonucleotides to the 5′
terminus of said tethered oligonucleotide, wherein said ligating is carried out in the presence of RNA ligase or ribozyme;
e) phosphorylating the 5′
terminus of said another oligonucleotide; and
f) repeating steps d) and e) until said polynucleotide is prepared.
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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.
14 Citations
35 Claims
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1. A method of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising the steps:
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a) providing a solid support;
b) providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of said oligonucleotides comprises the nucleotide sequence of said polynucleotide;
c) contacting said solid support with the 3′
terminus of a first oligonucleotide from said plurality of oligonucleotides to form a tethered oligonucleotide;
d) ligating the 3′
terminus of another oligonucleotide from said plurality of oligonucleotides to the 5′
terminus of said tethered oligonucleotide, wherein said ligating is carried out in the presence of RNA ligase or ribozyme;
e) phosphorylating the 5′
terminus of said another oligonucleotide; and
f) repeating steps d) and e) until said polynucleotide is prepared. - View Dependent Claims (2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 19)
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8. A method of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising the steps:
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a) providing a solid support;
b) providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of said oligonucleotides comprises the nucleotide sequence of said polynucleotide;
c) contacting said solid support with the 3′
terminus of a first oligonucleotide from said plurality of oligonucleotides to form a tethered oligonucleotide;
d) ligating the 3′
terminus of another oligonucleotide from said plurality of oligonucleotides to the 5′
terminus of said tethered oligonucleotide; and
e) repeating step d) until said polynucleotide is prepared, wherein prior to ligation of said tethered oligonucleotide to said another of said plurality of oligonucleotides, any tethered oligonucleotide with an unphosphorylated 5′
terminus is capped, and wherein said capping is carried out with an enzyme which oxidizes said 5′
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OH terminus of said unphosphorylated tethered oligonucleotide.- View Dependent Claims (9)
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10. A method of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising the steps:
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a) providing a solid support;
b) providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of said oligonucleotides comprises the nucleotide sequence of said polynucleotide;
c) contacting said solid support with the 3′
terminus of a first oligonucleotide from said plurality of oligonucleotides to form a tethered oligonucleotide;
d) ligating the 3′
terminus of another oligonucleotide from said plurality of oligonucleotides to the 5′
terminus of said tethered oligonucleotide; and
e) repeating step d) until said polynucleotide is prepared, wherein prior to ligation of said tethered oligonucleotide to said another of said plurality of oligonucleotides, any tethered oligonucleotide with an unphosphorylated 5′
terminus is capped, and wherein said capping is carried out with an enzyme which acylates said 5′
—
OH terminus of said unphosphorylated tethered oligonucleotide.- View Dependent Claims (11)
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20. A method of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising the steps:
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a) providing a solid support;
b) providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of said oligonucleotides comprises the nucleotide sequence of said polynucleotide;
c) contacting said solid support with the 5′
terminus of a first oligonucleotide from said plurality of oligonucleotides to form a tethered oligonucleotide;
d) ligating the 5′
terminus of another oligonucleotide from said plurality of oligonucleotides to the 3′
terminus of said tethered oligonucleotide, wherein the 3′
OH terminus of said another oligonucleotide is blocked by acylation; and
e) repeating step d) until said polynucleotide is prepared. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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Specification