In vitro capture of nucleic acids via modified oligonucleotides and magnetic beads

• US 20030032028A1
• Filed: 02/15/2002
• Published: 02/13/2003
• Est. Priority Date: 06/12/2001
• Status: Abandoned Application

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Goff, Stephen, Oeller, Paul, Dace, Gayle, Kimmerly, William
• Application Number:
  US10/077,275
• Publication Number:
  US 20030032028A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label

Abstract

A method is disclosed for capturing one or more target sequences utilizing modified, high stringency nucleic acids and strand displacement stategies. In this method, one or more modified, high stringency oligonucleotide (MHSO) conjugates are selected in which the conjugates are constructed from at least one modified nucleic acid, such as, for example, a locked nucleic acid (“LNA”) and a linking molecule. When incubated with a sample of nucleic acids, these MHSO conjugates capture the target sequence by selectively binding to complementary sequences of DNA or RNA, which may be either single or double stranded. Once bound, the resulting complexes constitute hybridized duplexes containing both a targeted sequence portion and a MHSO conjugate portion. The captured target sequences are separated from the sample by using a linking source that binds to the linking molecule of the hybridized duplex and extracting the linking source with the bound duplex from the sample.

Claims

1. An in vitro method of capturing one or more target sequences, wherein the method comprises the steps of:

providing one or more modified, high stringency oligonucleotide conjugates (“

MHSO”

) comprising a nucleotide sequence that is complementary to a target sequence and a linking molecule;

incubating a sample of nucleic acids comprising the target sequence to be captured with the MHSO conjugate under non-denaturing conditions, thereby forming one or more hybridized duplexes, wherein each duplex comprises a target sequence portion and a modified oligonucleotide conjugate portion;

contacting substantially all of the hybridized duplexes with a linking source, such that the linking molecule of each duplex that contacts the linking source forms a bond with the linking source; and

separating substantially all of the hybridized duplexes from the sample of nucleic acids by extracting the linking source from the sample.

2. The method of claim 1, further comprising the step of disassociating the targeted sequence portion of each of the hybridized duplexes from the linking source and modified oligonucleotide conjugate.

3. The method of claim 2, wherein the disassociating step further comprises incubating the hybridized duplexes with an alkaline buffer, such that the targeted sequence portion is disassociated from the modified oligonucleotide conjugate and the linking source.

4. The method of claim 3, wherein the alkaline buffer has a pH between 9 and 10.

5. The method of claim 1, wherein the incubating step comprises using reaction conditions at which substantially all of the target sequences form a strand displacing “

A”

helix with the modified oligonucleotide conjugates.

6. The method of claim 1, wherein the modified oligonucleotide is about 9-20 bases long.

7. The method of claim 1, wherein the modified oligonucleotide is 12 bases long.

8. The method of claim 1, wherein at least 50% of the nucleobases of the modifided oligonucleotide are modified nucleobases.

9. The method of claim 1, wherein at least 85% of the nucleobases of the modifided oligonucleotide are modified nucleobases.

10. The method of claim 1, wherein a 100% of the nucleobases of the modifided oligonucleotide are modified nucleobases.

11. The method of claim 8, wherein the modifided nucleobase is a locked nucleoside analogue (LNA).

13. The method of claim 1, wherein the incubation of the sample of nucleic acids comprising the target sequence with the MHSO conjugate is done in a low stringency buffer solution and at a temperature in the range of between about 65°

C. to about 80°

C.

14. The method according to claim 11, wherein the salt concentration of the buffer solution is in a range of between about 10 mM and 100 mM.

15. The method according to claim 1, wherein the method forms a new library enriched in the targeted sequence.

16. The method of claim 1, wherein the method forms a new library depleted in the targeted sequence.

17. The method of claim 1, wherein the target sequence portion of the hybridized duplex is a portion of the insert in a 3.5 kb clone.

18. The method of claim 1, further comprising the step of obtaining the sample from a plasmid library.

19. The method of claim 16, wherein at least one of the one or more plasmids is a circular plasmid.

20. The method of claim 1, further comprising the step of obtaining the sample from a double stranded DNA plasmid library.

21. The method of claim 1, further comprising the step of obtaining the sample from genomic DNA.

22. The method of claim 1, further comprising the step of obtaining the sample from RNA sequences.

23. The method of claim 1, wherein the linking molecule comprises biotin bound to a 5′

end of the modified oligonucleotide.

24. The method of claim 1, wherein the linking molecule comprises an antibody, biotin, immunoglobulin, or carbohydrate.

25. The method of claim 1, wherein the linking source comprises streptavidin-coated beads.

26. The method of claim 1, wherein the linking source comprises an antigen, avidin, protein A, or lectin.

27. The method of claim 1, wherein the separating step comprises using a magnet to extract the linking source and hybridized duplexes from the sample sequences.

29. The method of claim 1, wherein the sample of nucleic acids contains one or more nucleic acid molecules having a nucleotide sequence which comprises one or more target simple sequence repeats.

30. The method of claim 1, wherein the captured simple sequence repeat portion comprises 1, 2, 3, or 4 base repeats.

31. The method of claim 1, wherein the modified oligonucleotides comprise nucleotide sequences that are complementary to the target simple sequence repeats.

32. A library enriched with targeted simple sequence repeats, formed by the method of claim 1.

35. The hybridized duplex of claim 31, further comprising a linking source.

12. An in vitro method of capturing one or more target sequences, wherein the method comprises the steps of:

providing one or more LNA conjugates comprising a nucleotide sequence that is complementary to a target sequence and a linking molecule, wherein at least in one of the LNA conjugates 85% of the nucleobases are locked nucleoside analogues (LNAs);

incubating a sample of nucleic acids comprising the target sequence to be captured with the LNA conjugate in a low stringency buffer solution under non-denaturing conditions, thereby forming one or more hybridized duplexes, wherein each duplex comprises a target sequence portion and a modified oligonucleotide conjugate portion;

contacting substantially all of the hybridized duplexes with a linking source, such that the linking molecule of each duplex that contacts the linking source forms a bond with the linking source; and

separating substantially all of the hybridized duplexes from the sample of nucleic acids by extracting the linking source from the sample.

28. An in vitro method of capturing one or more target sequences, wherein the method comprises the steps of:

providing one or more LNA conjugates comprising a nucleotide sequence that is complementary to a target sequence and a linking molecule, wherein at least in one of the LNA conjugates 100% of the nucleobases are locked nucleoside analogues (LNAs);

incubating a sample of nucleic acids comprising the target sequence to be captured with the LNA conjugate in a low stringency buffer solution with a salt conentration of between 10 mM and 100 mM under non-denaturing conditions at a temperature of between about 65°

C. to about 80°

C., thereby forming one or more hybridized duplexes, wherein each duplex comprises a target sequence portion and a modified oligonucleotide conjugate portion;

contacting substantially all of the hybridized duplexes with a linking source, such that the linking molecule of each duplex that contacts the linking source forms a bond with the linking source; and

separating substantially all of the hybridized duplexes from the sample of nucleic acids by extracting the linking source from the sample.

33. An in vitro method of capturing one or more target simple sequence repeats, wherein the method comprises the steps of:

providing one or more modified oligonucleotide conjugates, wherein each of the modified oligonucleotide conjugates comprises at least one locked nucleic acid and a linking molecule;

incubating a sample of nucleic acids with the modified oligonucleotide conjugates, thereby forming one or more hybridized duplexes, wherein each duplex comprises a target simple sequence repeat portion and a modified oligonucleotide conjugate portion;

binding the linking molecule biotin on the modified oligonucleotides to streptavidin on coated magnetic beads, such that the magnetic beads are linked to the hybridization duplexes;

separating the hybridized duplexes from other materials with a magnet;

washing the hybridized duplexes;

incubating the hybridized duplexes with buffer of pH of about 9.5 such that the targeted simple sequence repeat dissociates from the modified oligonucleotide conjugate and the magnetic bead;

transforming the simple sequence repeats in E. coli; and

sequencing the transformed simple sequence repeats.

34. A hybridized duplex comprising a locked nucleic acid portion, and a simple sequence repeat portion.

36. A strand displacement method of capturing target nucleic acids, wherein the improvement comprises using one or more target nucleic acid molecules having a nucleotide sequence which comprises one or more simple sequence repeats.

37. A kit for capturing target simple sequence repeats, comprising:

one or more modified oligonucleotide conjugates, wherein each of the modified oligonucleotide conjugates comprises at least one locked nucleic acid and a linking molecule;

a hybridized duplex comprised of a simple sequence repeat portion and a modified oligonucleotide conjugate portion;

a linking source, wherein the linking source is attachable to the linking molecule of the modified oligonucleotide conjugate; and

a means for separating the hybridized duplexes from a sample of nucleic acids.