Methods of making di-tagged DNA libraries from DNA or RNA using double-tagged oligonucleotides

1. A method of making a di-tagged DNA library, comprising the steps of:

a) providing a double-tagged oligonucleotide (DTO), sequentially comprising a sequence tag A, a linker, and a sequence tag B;

b) providing a DNA or RNA fragment;

c) connecting said DTO to said DNA fragment or a cDNA fragment complementary to said DNA or RNA fragment to form a circular DNA-DTO molecule;

d) generating a linear DNA from said circular DNA-DTO molecule, having sequence tag A and tag B at its ends and collecting a population of said di-tagged DNAs to obtain a di-tagged DNA library.

2. The method of claim 1 , wherein said linker comprises a stopping sequence or a breaking site.

3. The method of claim 2 , wherein said stopping sequence comprises modified nucleotides or chemical moieties that can stop a DNA polymerization reaction.

4. The method of claim 3 , wherein said stopping sequence comprises abasic nucleotides.

5. The method of claim 3 , wherein said stopping sequence comprises nucleotide analogs that are sterically or electronically incompatible with active sites of DNA polymerases.

6. The method of claim 3 , wherein said stopping sequence comprises nucleoside analogs selected from 4-methylidole β-nucleoside, α-naphthalene nucleoside, and α-pyrene nucleoside.

7. The method of claim 3 , wherein said chemical moiety comprises DNA spacers selected from C3 phosphoramidite spacer, C9 triethylene glycol spacer, or C18 hexa-ethyleneglycol spacer.

8. The method of claim 2 , wherein said breaking site comprises a special region of said DTO that is susceptible to photo, enzymatic or chemical cleavage.

9. The method of claim 8 , wherein said breaking site comprises uracil nucleotides.

10. The method of claim 8 , wherein said breaking site comprises single stranded DNA or RNA.

11. The method of claim 8 , wherein said breaking site comprises a photo-cleavable nucleotide spacer.

12. The method of claim 8 , wherein said breaking site comprises modified nucleotides selected from 5-hydroxy-dCTP, 5-hydroxy-dUTP, 7-Deaza-7-nitro-dATP, and 7-Deaza-7-nitro-dGTP.

13. The method of claim 1 , wherein means of connecting said DTO and said DNA fragment or said cDNA is selected from DNA ligation, reverse transcription, or DNA polymerization.

14. The method of claim 1 , after step c), further comprising isolating circular DNA-DTO molecule by removing linear DNAs with exonuclease treatment.

15. The method of claim 1 , wherein means of generating said linear DNA from said circular DNA-DTO molecule is selected from PCR amplification and breaking at said breaking site.

16. The method of claim 1 , wherein said DTO is a double stranded DNA and a double stranded DNA fragment is provided in step b).

17. The method of claim 16 , wherein said linker of said DTO comprises a breaking site.

18. The method of claim 17 , comprising the steps of:

I. connecting said DTO to said DNA fragment to form said circular DNA-DTO molecule by ligation;

II. digesting linear DNAs using exonucleases;

III. generating said linear DNA with sequence tag A and tag B from said circular DNA-DTO molecule by breaking at said breaking site.

19. The method of claim 18 , wherein said DNA fragment comprises 3′ overhang of a stretch of same nucleotides, and said DTO comprises 3′ overhang of a stretch of complementary nucleotides.

20. The method of claim 18 , wherein said DNA fragment comprises a single 3′ A-overhang, and said DTO comprises a single 3′ T-overhang.

21. The method of claim 18 , wherein said DNA fragment comprises a single 3′ G-overhang, and said DTO comprises a single 3′ C-overhang.

22. The method of claim 18 , wherein said breaking site comprises uracil nucleotides; and wherein said breaking site is broken by treatment of uracil DNA glycosylase and endonuclease VIII.

23. The method of claim 18 , wherein said breaking site comprises a single stranded DNA/RNA sequence; and wherein said breaking site is broken by treatment of nuclease S1.

24. The method of claim 16 , comprising the steps of

I. connecting said dsDTO with said dsDNA to form a circular DNA-DTO molecule by ligation;

II. digesting linear DNA with exonuclease treatment;

III. randomly fragmenting said circular DNA-DTO molecule into smaller sequences,

IV. performing end repair and self-ligation of said smaller sequences to form a smaller circular DNA.

25. The method of claim 24 , wherein said DTO comprises a stopping sequence.

26. The method of claim 25 , wherein said stopping sequence comprises abasic nucleotides.

27. The method of claim 24 , further comprising PCR amplification with said smaller circular DNA to generate a linear DNA with sequence tag A and tag B.

28. The method of claim 24 , wherein said linker of said DTO comprises a breaking site.

29. The method of claim 28 , further comprising removing linear DNA by exonuclease treatment, inactivating and removing exonuclease, and breaking said smaller circular DNA at the breaking site to generate a linear DNA with sequence tag A and tag B.

30. The method of claim 24 , wherein said DTO comprises a capture domain.

31. The method of claim 1 , wherein said DTO is a double-stranded DNA with a nick on each of its strands and a double stranded DNA fragment is provided in step b).

32. The method of claim 31 , comprising the steps of:

I. connecting said DTO to said DNA fragment to form a large circular DNA-DTO by ligation;

II. performing nick translation to elongate 3′ end of each said nick site;

III. using T7 exonuclease to create a single stranded DNA gap and S1 nuclease to cleave said single stranded DNA gap;

IV. performing end repair and self-ligation to form a smaller circular DNA-DTO.

33. The method of claim 32 , wherein said DTO comprises a breaking site.

34. The method of claim 33 , further comprising breaking said smaller circular DNA at said breaking site to generate a linear DNA with sequence tag A and tag B.

35. The method of claim 34 , further comprising PCR amplification of said linear DNA with sequence tag A and tag B.

36. The method of claim 32 , wherein said DTO comprises a stopping sequence.

37. The method of claim 36 , wherein said stopping sequence comprises abasic nucleotides.

38. The method of claim 36 , further comprising PCR amplification of said smaller circular DNA-DTO to generate a linear DNA with sequence tag A and tag B.

39. The method of claim 1 , wherein a single stranded RNA or DNA fragment is provided in step b), and said DTO comprises a single-stranded DNA, a stopping sequence, and a priming sequence.

40. The method of claim 39 , comprising the steps of:

a) synthesizing a cDNA-DTO using said DTO as a primer and said RNA or DNA fragment as a template;

b) ligating 3′ and 5′ end of cDNA-DTO to form a circular DNA-DTO molecule;

c) performing a PCR amplification to generate a linear DNA with sequence tag A and tag B at its ends.

41. The method of claim 40 , wherein said priming sequence comprises a random sequence.

42. The method of claim 40 , said priming sequence comprises a poly(dT) tail.

43. The method of claim 40 , wherein a poly(A) tail is added to said RNA fragment.

44. The method of claim 40 , wherein said poly(A) tail is incorporated with at least one biotin-AMP.

45. The method of claim 40 , wherein said stopping sequence comprises abasic nucleotides.