Use of phosphorothiolate polynucleotides in ligating nucleic acids
First Claim
1. A method of non-enzymatic ligation of a nucleic acid, comprising contacting a polynucleotide-3′
- phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-3′
phosphorothiolate and said acceptor polynucleotide.
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Abstract
The invention provides a method of nonenzymatic ligation of a nucleic acid. The method consists of contacting a polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between the polynucleotide-3′ phosphorothiolate and the acceptor polynucleotide. The invention also provides methods of molecular cloning. In one embodiment, the method consists of contacting an insert comprising a polynucleotide-3′ phosphorothiolate with an acceptor vector under conditions that allow formation of a phosphodiester bond between the insert and the acceptor vector to generate a vector comprising an insert polynucleotide. The invention further provides a compound consisting of a polynucleotide-3′ phosphorothiolate and a kit containing a polynucleotide-3′ phosphorothiolate. Also provided is a method of ligating a nucleic acid using a non-sequence specific topoisomerase.
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Citations
56 Claims
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1. A method of non-enzymatic ligation of a nucleic acid, comprising contacting a polynucleotide-3′
- phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-3′
phosphorothiolate and said acceptor polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7)
- phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-3′
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8. A method of molecular cloning comprising, contacting an insert comprising a polynucleotide-3′
- phosphorothiolate with an acceptor vector under conditions that allow formation of a phosphodiester bond between said insert and said acceptor vector to generate a vector comprising an insert polynucleotide.
- View Dependent Claims (9, 10, 11)
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12. A method of molecular cloning comprising, contacting a vector comprising a polynucleotide-3′
- phosphorothiolate with an acceptor polynucleotide, under conditions that allow formation of a phosphodiester bond between said vector and said acceptor polynucleotide to generate a vector comprising said acceptor polynucleotide.
- View Dependent Claims (13, 14, 15, 16)
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17. A kit, comprising:
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(a) a polynucleotide-3′
phosphorothiolate; and
(b) a buffer in an aqueous solution. - View Dependent Claims (18, 19, 20, 21, 22)
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23. A kit, comprising:
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(a) a polynucleotide-3′
phosphorothiolate precursor; and
(b) an activator. - View Dependent Claims (24, 25, 26, 27, 28)
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29. A method of ligating a nucleic acid, comprising contacting a polynucleotide-5′
- phosphorothiolate with a non-sequence specific topoisomerase, or a fragment or modification thereof, and an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-5′
phosphorothiolate and said acceptor polynucleotide, with the proviso that said polynucleotide-5′
phosphorothiolate does not contain the nucleotide sequence G(C/T)CCTT (SEQ ID NO;
5). - View Dependent Claims (30, 31, 32, 33, 34, 35)
- phosphorothiolate with a non-sequence specific topoisomerase, or a fragment or modification thereof, and an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-5′
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36. A kit, comprising:
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(a) a polynucleotide-5′
phosphorothiolate, with the proviso that said polynucleotide-5′
phosphorothiolate does not contain a nucleotide sequence selected from the group of SEQ ID NO;
5, SEQ ID NO;
6 or SEQ ID NO;
7;
(b) a non-sequence specific topoisomerase, or fragment or modification thereof having topoisomerase activity. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43)
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44. A composition comprising,
(a) a polynucleotide-5′ - phosphorothiolate, with the proviso that said polynucleotide-5′
phosphorothiolate does not contain a nucleotide sequence selected from the group of SEQ ID NO;
5, SEQ ID NO;
6 or SEQ ID NO;
7; and
(b) a non-sequence specific topoisomerase, or fragment or modification thereof having topoisomerase activity. - View Dependent Claims (45, 46, 47, 48, 49, 50, 51)
- phosphorothiolate, with the proviso that said polynucleotide-5′
- 52. A compound of the formula:
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56. A compound of the formula:
Specification