US2012322109A1PendingUtilityA1
Covalent Joining of DNA Strands to RNA Strands Catalyzed by Vaccinia Topoisomerase
Est. expiryJun 12, 2017(expired)· nominal 20-yr term from priority
C07H 21/04C12N 15/1006C12N 9/90C12Y 599/01002C12N 15/1096C07H 21/02
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Claims
Abstract
The present invention provides a method of covalently joining a DNA strand to an RNA strand, a method of tagging a 5′ end of an RNA molecule, a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase, a method of tagging a 5′ end of an mRNA, and a method of isolating and cloning full-length gene sequences using capped mRNA after subtraction of non-capped RNA.
Claims
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10 . A topoisomerase-DNA intermediate molecule comprising one or more 5′ single-strand tails.
11 . The topoisomerase-DNA intermediate molecule of claim 10 , wherein the 5′ single-strand tail comprises a specific sequence.
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18 . A DNA-RNA molecule covalently joined by topoisomerase catalysis.
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23 . A covalently joined DNA-RNA molecule having a labeled 5′ end.
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38 . A 5′ end tagged RNA molecule.
39 . The 5′ end tagged RNA molecule of claim 38 , wherein the tag is a DNA sequence.
40 . The 5′ end tagged RNA molecule of claim 39 , further comprising a 5′ end label.
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44 . A DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase.
45 . A method of obtaining full-length gene sequences comprising:
(a) isolating full-length mRNA: (b) attaching a DNA tag sequence to the isolated mRNA; and (c) synthesizing cDNA using the tagged mRNA as a template.
46 . A method of claim 45 , wherein the mRNA is isolated by employing an affinity purification material.
47 . A method of claim 46 , wherein the mRNA to be isolated comprises an affinity purification tagged cap structure.
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67 . An isolated full-length gene sequence prepared by the method of claim 45 .
68 . A nucleic acid construct comprising an isolated fulllength gene sequence prepared of the method of claim 45 and an expression vector.
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79 . A method of obtaining full-length gene sequences 5 comprising:
(a) isolating full-length mRNA by employing an affinity purification material; (b) decapping and dephosphorylating the isolated mRNA; (c) attaching a DNA tag sequence to the decapped, dephosphorylated mRNA, wherein the tag sequence comprises the sequence shown in FIG. 11 and is attached by vaccinia DNA topoisomerase; (d) synthesizing cDNA using the tagged mRNA as a template; (e) amplifying the synthesized cDNA, wherein the amplification primers comprise an anti-coding sequence of the tag sequence (5′) and a gene specific sequence (3′); and (f) inserting the amplified cDNA into an expression vector.Join the waitlist — get patent alerts
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