Recovery of original template
First Claim
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1. A hairpin nucleic acid, having the following characteristics:
- (a) being self-complementary; and
(b) having a first restriction site for a nicking endonuclease, said restriction site comprising a recognition sequence and a cleavage site, wherein said recognition sequence is situated so that said cleavage site is before, at, or beyond the 3′
end of the hairpin nucleic acid.
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Abstract
The present invention relates to methods for regenerating a single-stranded nucleic acid template following its conversion to a double-stranded product, e.g., during a polymerase reaction, and also to regenerating a hairpin or anchoring sequence by removal of the template and its synthesized complement, by design of enzyme restriction sites into the hairpin or anchoring sequence.
29 Citations
26 Claims
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1. A hairpin nucleic acid, having the following characteristics:
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(a) being self-complementary; and
(b) having a first restriction site for a nicking endonuclease, said restriction site comprising a recognition sequence and a cleavage site, wherein said recognition sequence is situated so that said cleavage site is before, at, or beyond the 3′
end of the hairpin nucleic acid. - View Dependent Claims (2, 3, 6, 7, 8, 9, 10, 11, 12, 13)
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4. A method for recovering a single-stranded template nucleic acid, the method comprising:
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(a) providing a single-stranded template nucleic acid attached to the 5′
end of a hairpin nucleic acid, wherein the hairpin nucleic acid is self-complementary and has a first restriction site for a nicking endonuclease, said restriction site comprising a recognition sequence and a cleavage site, wherein said recognition sequence is situated so that said cleavage site is before, at, or beyond the 3′
end of the hairpin nucleic acid, and wherein said hairpin nucleic acid is a self-hybrid, and wherein a nucleic acid strand complementary to the template nucleic acid is attached to the 3′
end of the hairpin nucleic acid;
(b) contacting the hairpin nucleic acid with said nicking endonuclease, under conditions where the nicking endonuclease cleaves before, at or beyond the 3′
end of the hairpin nucleic acid, thereby providing a nicked hairpin-template-complement nucleic acid complex; and
(c) subjecting the nicked hairpin-template-complement nucleic acid complex to conditions whereby the nucleic acid strand complementary to the template nucleic acid dissociates from the template nucleic acid;
thereby recovering the single-stranded template nucleic acid. - View Dependent Claims (5)
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14. A double-stranded nucleic acid anchor, having the following characteristics:
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(a) having a first end and a second end; and
(b) having a first restriction site for a nicking endonuclease, said restriction site comprising a recognition sequence and a cleavage site, wherein said recognition sequence is situated so that said cleavage site is located before, at, or beyond the 3′
end of the first end of the double-stranded nucleic acid anchor. - View Dependent Claims (15, 16, 19, 20, 21, 22, 23, 24, 25, 26)
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17. A method for recovering a single-stranded template nucleic acid, the method comprising:
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(a) providing a single-stranded template nucleic acid attached to a double-stranded nucleic acid anchor, and wherein a nucleic acid strand complementary to the template nucleic acid is attached to the double-stranded nucleic acid anchor, and wherein the double-stranded nucleic acid anchor;
(i) has a first end and a second end; and
(ii) has a first restriction site for a nicking endonuclease, said restriction site comprising a recognition sequence and a cleavage site, wherein said cleavage site is situated so that said cleavage site is before, at, or beyond the 3′
end of the first end of the double-stranded nucleic acid anchor;
wherein the single-stranded template nucleic acid is attached to the 5′
end of the first end of the double-stranded nucleic acid anchor, and wherein the nucleic acid strand complementary to the template nucleic acid is attached to the 3′
end of the first end of the double-stranded nucleic acid anchor;
(b) contacting the double-stranded nucleic acid anchor with said nicking endonuclease, under conditions where the nicking endonuclease cleaves before, at, or beyond the 3′
end of the first end of the double-stranded nucleic acid anchor, thereby providing a nicked anchor-template-complement nucleic acid complex; and
(c) subjecting the nicked anchor-template-complement nucleic acid complex to conditions whereby the nucleic acid strand complementary to the template nucleic acid dissociates from the template nucleic acid;
thereby recovering the single-stranded template nucleic acid. - View Dependent Claims (18)
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Specification