Claims
- 1. A method of nucleic acid amplification, comprising:
(a) contacting a linear amplification molecule to a nucleic acid template under conditions that support specific hybridization, (b) adding sequentially to said linear amplification molecule at the end hybridized to said template one or more nucleotides complementary to corresponding nucleotides present in said template,
whereby an extended linear amplification molecule is formed, (c) incubating said extended linear amplification molecule under conditions that support its specific intramolecular hybridization,
whereby a double-stranded intramolecular hybridization region is formed, wherein one of the stands comprises nucleotides newly added in step (b), (d) cleaving said extended linear amplification molecule at a predetermined site,
whereby a first fragment and a second fragment of said extended linear amplification molecule are formed; and whereby said first fragment contacts said second fragment along said intramolecular hybridization region; and (e) adding sequentially to said first fragment at the end newly created by said cleavage step one or more nucleotides complementary to corresponding nucleotides present in said second fragment,
whereby an extended first fragment is formed.
- 2. The method of claim 1 further comprising the step, after step (e), of amplifying exponentially a sequence present in said extended first fragment.
- 3. The method of claim 2, wherein said step of amplifying exponentially is effected using the polymerase chain reaction.
- 4. The method of claim 1 further comprising the step of eliminating unhybridized linear amplification molecules.
- 5. The method of claim 4, wherein said step of eliminating unhybridized linear amplification molecules is effected using an exonuclease.
- 6. The method of claim 1, wherein said linear amplification molecule comprises a uracil base.
- 7. The method of claim 6, wherein step (b) is effected using a DNA polymerase that stalls at a uracil base.
- 8. The method of claim 1, wherein said extended first fragment of step (e) comprises an affinity capture moiety.
- 9. The method of claim 8, wherein said affinity capture moiety is a biotinylated nucleotide.
- 10. The method of claim 9, further comprising the step of contacting said extended first fragment with a biotin-binding moiety.
- 11. The method of claim 10, wherein said biotin-binding moiety is selected from among the group consisting of: avidin, streptavidin, biotin-specific antibody.
- 12. The method of claim 1, wherein said linear amplification molecule comprises an oligonucleotide.
- 13. The method of claim 12, wherein said oligonucleotide comprises deoxyribonucleic acid.
- 14. The method of claim 1, wherein said linear amplification molecule comprises a fluorescent moiety.
- 15. The method of claim 1, wherein said template is selected from among the group consisting of: DNA, plasmid DNA, genomic DNA, viral DNA, bacterial DNA, nuclear DNA, mitochondrial DNA, cellular DNA, RNA, mRNA, hnRNA, rRNA, viral genomic RNA.
- 16. The method of claim 1, wherein said steps of adding nucleotides sequentially is effected using a DNA polymerase.
- 17. The method of claim 1, wherein said step of cleavage is effected using one or more enzymes.
- 18. The method of claim 1, wherein said predetermined site of cleavage is an apurinic site.
- 19. The method of claim 1, wherein said step of cleavage is effected using a chemical agent.
- 20. The method of claim 2, further comprising the step of analyzing said exponentially amplified sequence.
- 21. A linear molecule for nucleic acid amplification, comprising:
a) a first primer region substantially similar to a first primer sequence, b) a first homology region substantially similar to the sequence of a first homology region of a nucleic acid, c) a cleavable site, d) a second primer region substantially similar to a second primer sequence; and e) a second homology region substantially similar to the complement of the sequence of a second homology region of the nucleic acid,
wherein said first and second homology regions of the nucleic acid reside on the same single-strand of nucleic acid; and wherein said first homology region of the nucleic acid is proximal to the 5′ end of the strand and said second homology region is distal to the 5′ end of the strand.
- 22. The linear amplification molecule of claim 21 further comprising a barcode sequence.
- 23. The linear amplification molecule claim 22, wherein said barcode sequence is between the first primer region and the first homology region.
- 24. The linear amplification molecule of claim 23, wherein said barcode sequence is between the second primer region and the second homology region.
- 25. The linear amplification molecule of claim 21, further comprising a fluorescent moiety.
- 26. The linear amplification molecule of claim 21 further comprising an affinity capture moiety.
- 27. The linear amplification molecule of claim 26, wherein said affinity capture moiety is biotin.
- 28. The linear amplification molecule of claim 21, wherein said linear amplification molecule is an oligonucleotide.
- 29. The linear amplification molecule of claim 28, wherein said oligonucleotide comprises deoxyribonucleic acid.
- 30. The linear amplification molecule of claim 29, wherein said oligonucleotide comprises a uracil base.
- 31. The linear amplification molecule of claim 21, wherein said cleavable site is capable of specific cleavage with an enzyme.
- 32. The linear amplification molecule of claim 31, wherein said cleavage site is capable of forming an apurinic site.
- 33. The linear amplification molecule of claim 21, wherein said cleavable site is capable of specific cleavage with a chemical agent.
- 34. A method of nucleic acid amplification, comprising:
(a) contacting a linear amplification molecule to a nucleic acid template under conditions that support specific hybridization,
adding sequentially to said linear amplification molecule at the end hybridized to said template one or more nucleotides complementary to corresponding nucleotides present in said template, whereby an extended linear amplification molecule is formed, (c) incubating said extended linear amplification molecule under conditions that support its specific intramolecular hybridization,
whereby an intramolecular hybridization region is formed, (d) removing one or more nucleotides that were newly added in step (b) and that are not specifically hybridized in said intramolecular hybridization region from the end of said extended linear amplification molecule; and
adding sequentially to said extended linear amplification molecule at the end hybridized in said intramolecular hybridization region one or more nucleotides complementary to corresponding nucleotides present in said linear amplification molecule.
- 35. The method of claim 34 further comprising the step, after step (e), of amplifying exponentially said extended linear amplification molecule.
- 36. A linear molecule for nucleic acid amplification, comprising:
a) a primer region substantially similar to a primer sequence, b) a first homology region substantially similar to the sequence of a first homology region of a nucleic acid; and c) a second homology region substantially similar to the complement of the sequence of a second homology region of the nucleic acid,
wherein said first and second homology regions of the nucleic acid reside on the same single-strand of nucleic acid; and wherein said first homology region of the nucleic acid is proximal to the 5′ end of the strand and said second homology region of the nucleic acid is distal to the 5′ end of the strand.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Application Ser. No. 60/331,693, filed Nov. 19, 2001, the disclosure of which is incorporated herein by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60331693 |
Nov 2001 |
US |