Claims
- 1. A double stranded topoisomerase-adapted linker comprising a duplex oligonucleotide linker, wherein a first oligonucleotide is annealed to a second oligonucleotide that is phosphorylated at the 5′ end thereof, and wherein the linker further comprises a compatible site-specific topoisomerase enzyme covalently attached to the first oligonucleotide at a single base 3′ T overhang.
- 2. A topoisomerase-adapted linker according to claim 1, wherein the site-specific topoisomerase is a Topoisomerase I.
- 3. A topoisomerase-adapted linker according to claim 1, wherein the site-specific topoisomerase is a Vaccinia topoisomerase and the cleavage site comprises SEQ ID NO: 4.
- 4. A topoisomerase-adapted linker according to claim 1, wherein the first oligonucleotide comprises an oligonucleotide sequence according to SEQ ID NO: 9 and the second oligonucleotide is complementary to the first nucleotide and is phosphorylated at the 5′ end thereof.
- 5. A topoisomerase linker according to claim 4, wherein the first oligonucleotide comprises an oligonucleotide sequence according to SEQ ID NO: 9 and the second oligonucleotide comprises an oligonucleotide sequence according to SEQ ID NO: 10.
- 6. A topoisomerase linker according to claim 1, wherein the first oligonucleotide is modified by attachment of biotin at the 5′ end thereof.
- 7. A topoisomerase linker according to claim 1, wherein the second oligonucleotide further includes a poly-A tail at the 3′ end thereof.
- 8. A topoisomerase linker-specific oligonucleotide having a sequence according to SEQ ID NO: 11.
- 9. A topoisomerase linker-specific oligonucleotide having a sequence according to SEQ ID NO: 12.
- 10. A method for isolating a target polynucleotide having a segment of unknown sequence when sequence of 3′ nucleic acid is known, said method comprising:
(a) cutting the target polynucleotide with a restriction endonuclease that leaves a 3′ overhang of at least 2 bases to obtain an overhang-digested polynucleotide segment, (b) dephosphorylating the 5′ ends of the overhang-digested polynucleotide segment, (c) performing a Taq polymerase mediated primer extension of the 5′-dephosphorylated overhang-digested polynucleotide segment using a sequence-specific primer to create a duplex extension product having at the 3′ ends a single A base overhang, (d) incubating the duplex extension product with a topoisomerase-adapted linker according to claim 1 so as to selectively attach the duplex DNA of the linker to the extension product to form a linked extension product, and (e) isolating a second extension product produced by amplifying the linked extension product using the sequence-specific primer and a topoisomerase linker-specific primer.
- 11. The method according to claim 10, wherein the method further comprises obtaining the sequence of the isolated second extension product.
- 12. The method according to claim 11, wherein the second extension product is FPLC gel-purified prior to sequencing.
- 13. The method according to claim 11, wherein the sequence is obtained without cloning of the second extension product into a vector.
- 14. The method according to claim 10, wherein the Taq polymerase is Taq I polymerase.
- 15. The method according to claim 10, wherein the sequence-specific topoisomerase is a Vaccinia topoisomerase enzyme.
- 16. The method according to claim 10, wherein the topoisomerase linker comprises a covalently attached Vaccinia topoisomerase I and binds only to DNA containing a single 3′ A base overhang.
- 17. The method according to claim 10, wherein the topoisomerase linker is the topoisomerase linker according to claim 4 and the linker-specific primer is a nucleotide having the sequence of SEQ ID NO: 11.
- 18. The method according to claim 10, wherein the topoisomerase linker is the topoisomerase linker according to claim 4 and the linker-specific primer is a nucleotide having the sequence of SEQ ID NO: 12.
- 19. The method according to claim 10, wherein the target nucleotide sequence is DNA.
- 20. The method according to claim 10, wherein the target nucleotide sequence is RNA.
- 21. A method for contiguous sequencing of genomic DNA having a segment of unknown sequence in a vector, said method comprising:
(a) cutting the nucleotide sequence of the vector with a restriction endonuclease that leaves a 3′ overhang of at least 2 bases to obtain an overhang-digested polynucleotide segment that contains an oligonucleotide of unknown sequence, (b) dephosphorylating the 5′ ends of the overhang-digested polynucleotide segment, (c) performing a Taq polymerase mediated primer extension of the 5′-dephosphorylated overhang-digested polynucleotide segment using a vector sequence-specific primer to create a duplex extension product having at the 3′ ends a single A base overhang, (d) incubating the duplex extension product with a topoisomerase-adapted linker according to claim 1 so as to selectively join the duplex DNA contained in the linker to the extension product to form a linked extension product, and (e) isolating a second extension product produced by amplifying the linked extension product using the vector sequence-specific primer and a topoisomerase linker-specific primer.
- 22. The method according to claim 21 further comprising obtaining a nested extension product by nested Taq-mediated PCR amplification of the second extension product using a set of sequence-specific and topoisomerase linker-specific primers that are internal to the primers used in step (d) above.
- 23. The method according to claim 21, wherein the overhang-digested DNA is from about 1 kilobase to about 50 kilobases in length.
- 24. The method according to claim 21, wherein the vector is an artificial chromosome vector.
- 25. The method according to claim 24, wherein the artificial chromosome vector is a bacterial artificial chromosome vector.
- 26. The method according to claim 24, wherein the artificial chromosome vector is a yeast artificial chromosome vector.
- 27. The method according to claim 26, wherein the yeast artificial chromosome vector is a Lambda vector.
- 28. The method according to claim 27, wherein the restriction endonuclease is PstI.
- 29. The method according to claim 21, wherein the topoisomerase linker is a topoisomerase linker according to claim 4, and wherein the topoisomerase linker-specific primer has a nucleic acid sequence according to SEQ ID NO: 11.
- 30. The method according to claim 21, wherein the method further comprises sequencing the isolated second extension product.
- 31. A kit for sequencing contiguous sequencing of genomic DNA that is cloned in a vector, said kit comprising:
(a) topoisomerase-adapted linker according to claim 1, and (b) a topoisomerase linker-specific oligonucleotide, wherein the linker-specific oligonucleotide hybridizes with the duplex DNA of the linker under stringent conditions.
- 32. The kit according to claim 31, further comprising:
(c) a PstI-cut Lambda vector and (d) a nucleotide primer that anneals to the PstI-cut Lambda vector, wherein the genomic DNA is cloned in the Lambda vector.
- 33. A method for amplifying by the polymerase chain reaction (PCR) a portion of a target nucleic acid sequence, said method comprising:
(a) cutting the DNA with a restriction endonuclease that leaves a 3′ overhang of at least 2 bases to obtain an overhang-digested DNA, (b) removing the 5′ phosphate groups from the overhang-digested DNA, (c) performing a Taq polymerase-mediated primer extension of the overhang-digested DNA using a sequence-specific primer to create a duplex extension product having at the 3′ ends a single A base overhang, (d) incubating the duplex extension product with a topoisomerase-adapted linker according to claim 1 so as to covalently attach the duplex DNA of the linker to the extension product to form a linked extension product, and (e) performing PCR amplification of the linked extension product using the sequence-specific primer and a topoisomerase linker-specific primer.
- 34. The method according to claim 33, wherein said amplification is under conditions of sufficient stringency so that specific amplification occurs.
- 35. The method of claim 34, further comprising sequencing the amplification product of step (e), thereby determining the sequence of a portion of said target nucleic acid sequence.
- 36. The method of claim 33, wherein the target nucleic acid is DNA.
- 37. The method of claim 33, wherein the target nucleic acid is RNA.
- 38. The method of claim 35, wherein the target nucleic acid is DNA.
- 39. The method of claim 35, wherein the target nucleic acid is RNA.
Parent Case Info
[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Ser. No. 60/184,858, filed Feb. 25, 2000, the entire contents of which is incorporated herein by reference.
Government Interests
[0002] This invention was made with support under Grant No. 4 R44 CA 80224 from the National Institutes of Health, U.S. Department of Health and Human Services. Accordingly, the United States Government has certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60184858 |
Feb 2000 |
US |