Claims
- 1. A method of appending at least a first oligonucleotide directly to a nucleic acid template, the method comprising:
concurrently annealing the template and at least a first oligonucleotide to at least a first probe,
wherein said probe includes at least a first template complementarity region and at least a first oligo positioning region directly adjacent thereto, the nucleotide of the template complementarity region and the nucleotide of the oligo positioning region that are directly adjacent within said probe being first junctional nucleotides that define a first probe junction therebetween, and wherein said oligonucleotide includes a terminal region that is complementary to the first oligo positioning region of said probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the probe's first oligo positioning region; creating a first ligatable free end at the template nucleotide that is annealed to the junctional nucleotide of the probe's first template complementarity region; and then ligating said first oligonucleotide to said template first free end.
- 2. The method of claim 1 wherein said probe includes a second oligo positioning region directly adjacent to the first template complementarity region, the nucleotide of the template complementarity region and the nucleotide of the second oligo positioning region that are directly adjacent within said probe being second junctional nucleotides that define a second probe junction therebetween.
- 3. The method of claim 2, wherein a second oligonucleotide is appended to said template, by:
annealing a second oligonucleotide to the first probe concurrently with annealing of said template to said probe, wherein said second oligonucleotide includes a terminal region that is complementary to the second oligo positioning region of said probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the probe's second oligo positioning region; creating a second ligatable free end at the template nucleotide that is annealed to the second junctional nucleotide of the probe's first template complementarity region; and then ligating said second oligonucleotide to said template second free end.
- 4. The method of claim 3, wherein each of said ligatable free ends is created by removing template regions that are noncomplementary to said probe first complementarity region.
- 5. The method of claim 4, wherein said template noncomplementary regions are removed by exonucleolytic digestion.
- 6. The method of claim 5, wherein said template noncomplementary regions are removed by digestion with Exonuclease VII or a combination of Exonuclease T and rec Jf.
- 7. The method of claim 4, wherein said template noncomplementary regions are removed by digestion with a single-strand specific endonuclease.
- 8. The method of claim 7, wherein said endonuclease is mung bean nuclease.
- 9. The method of claim 3, further comprising the step, after ligating, of:
separating said template from said probe and said oligonucleotides.
- 10. The method of claim 9, wherein said probe further comprises means for separating said probe from said template.
- 11. The method of claim 10, wherein said separating means are a plurality of incorporated deoxyuridine residues.
- 12. The method of claim 11, wherein said separating step comprises cleaving said probe at said deoxyuridine residues into a plurality of probe fragments, and then size separating said template from said probe fragments and from said oligonucleotides
- 13. The method of claim 10, wherein said separating means are one or more capture moieties.
- 14. The method of claim 9, further comprising the step, after separating said template from said probe and said oligonucleotides, of:
amplifying a region of said template between said first and second appended oligonucleotides.
- 15. The method of claim 14, wherein said first oligonucleotide has a first priming site and said second oligonucleotide has a second priming site, and said amplifying is performed by priming polymerization at said first priming site and said second priming site.
- 16. The method of claim 15, wherein said template region is amplified by PCR.
- 17. The method of claim 3, wherein said template is derived from cDNA.
- 18. The method of claim 3, wherein said template is derived from genomic DNA.
- 19. The method of claim 18, wherein said genomic DNA is from a single individual.
- 20. The method of claim 18, wherein said genomic DNA pooled from a plurality of individuals.
- 21. The method of claim 18, wherein said genomic DNA is derived from a eukaryote.
- 22. The method of claim 14, wherein at least one of said first and second oligonucleotides comprises a bar code sequence.
- 23. A method of appending at least a first oligonucleotide directly to each of a plurality of nucleic acid templates of distinct sequence in a single reaction, the method comprising:
concurrently annealing each template and a respective first oligonucleotide to a respective one of a plurality of probes within a single reaction mixture,
wherein each probe includes at least a first region of complementarity to a respective one of said templates and at least a first oligo positioning region directly adjacent thereto, the nucleotide of the template complementarity region and the nucleotide of the oligo positioning region that are directly adjacent within said probe being first junctional nucleotides that define a first probe junction therebetween, and wherein each said first oligonucleotide includes a terminal region that is complementary to the first oligo positioning region of a respective probe, the terminal nucleotide of said oligonucleotide region being annealed to the first junctional nucleotide of the probe's first oligo positioning region; creating a first ligatable free end at the nucleotide of each template that is annealed to the junctional nucleotide of its respective probe's first template complementarity region; and then ligating each said first oligonucleotide to its respective template first free end.
- 24. The method of claim 23 wherein each said probe includes a second oligo positioning region directly adjacent to the first template complementarity region, the nucleotide of the template complementarity region and the nucleotide of the second oligo positioning region that are directly adjacent within said probe being second junctional nucleotides that define a second probe junction therebetween.
- 25. The method of claim 24, wherein a second oligonucleotide is appended to each of said plurality of templates of distinct sequence by:
annealing a respective second oligonucleotide to each probe concurrently with annealing of said template to said probe, wherein said second oligonucleotide includes a terminal region that is complementary to the second oligo positioning region of its respective probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the probe's second oligo positioning region; creating a second ligatable free end at the template nucleotide that is annealed to the second junctional nucleotide of the probe's first template complementarity region; and then ligating said second oligonucleotide to said template second free end.
- 26. The method of claim 25, wherein each of said first oligonucleotides includes a priming sequence that is common thereamong.
- 27. The method of claim 26, wherein each of said second oligonucleotides includes a priming sequence that is common thereamong.
- 28. The method of claim 25, wherein each of said ligatable free ends is created by removing template regions that are noncomplementary to said probe first complementarity region.
- 29. The method of claim 28, wherein said template noncomplementary regions are removed by exonucleolytic digestion.
- 30. The method of claim 27, further comprising the step, after ligating, of:
separating said templates from said probes and said oligonucleotides.
- 31. The method of claim 30, further comprising the step, after separating said templates from said probes and said oligonucleotides, of:
concurrently amplifying a region of each said template.
- 32. The method of claim 31, wherein said amplifying is performed by priming polymerization at said first common priming site and said second common priming site.
- 33. The method of claim 32, wherein said template region is amplified by PCR.
- 34. The method of claim 33, wherein said plurality includes at least 10 templates of distinct sequence.
- 35. The method of claim 34, wherein said plurality includes at least 100 templates of distinct sequence.
- 36. The method of claim 35, wherein said plurality includes at least 1000 templates of distinct sequence.
- 37. The method of claim 27, wherein at least one of said first and second oligonucleotides comprises a bar code sequence.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional application serial 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 |