Insertion of sequence elements into nucleic acids

Patent metadata

Field	Value
Publication number	US-8932831-B2
Application number	US-74455307-A
Country	US
Kind code	B2
Filing date	May 4, 2007
Priority date	May 5, 2006
Publication date	Jan 13, 2015
Grant date	Jan 13, 2015

Abstract

The present invention concerns a method for inserting one or more tag sequences into a nucleic acid characterized by the following steps: (a) preparation of a template nucleic acid; (b) hybridization of at least one anchor sequence of at least one anchor oligonucleotide with one sequence section of the template nucleic acid; and (c) synthesization of a new strand of nucleic acid, which is partially complementary to the template nucleic acid and which contains a sequence complementary to the non-hybridized portion of the anchor oligonucleotide, e.g. to at least one tag sequence, on its 3′ end.

First claim

What is claimed is:

1. A method for random insertion of a tag sequence into a nucleic acid comprising:
in a first strand synthesis reaction,
(a) providing a template nucleic acid and a primer,
(b) hybridizing an anchor oligonucleotide to the template nucleic acid,
wherein the anchor oligonucleotide comprises an anchor sequence and a template tag sequence,
wherein the anchor sequence is a random sequence,
and
wherein the template tag sequence is a non-hybridized portion of the anchor oligonucleotide; and

(c) extending the primer of step (a), along the template nucleic acid to form a new strand of nucleic acid,
wherein a portion of the new strand of nucleic acid comprises a sequence complementary to the template nucleic acid, and
wherein a portion of the resulting new strand of nucleic acid comprises a sequence complementary to the template tag sequence of the anchor oligonucleotide
thereby randomly inserting a sequence complementary to the template tag sequence of the anchor oligonucleotide into a new strand of nucleic acid.

2. The method of claim 1 , wherein the anchor sequence of the anchor oligonucleotide is located at the 3′ region and the template tag sequence of the anchor oligonucleotide is located in the 5′ region of the anchor oligonucleotide.

3. The method of claim 1 , characterized in that the template tag sequence contains at least one functional sequence.

4. The method of claim 2 , characterized in that the template tag sequence contains at least one functional sequence.

5. The method of claim 1 , wherein a polymerase with slight or no strand displacement activity is used in Step (c).

6. The method of claim 2 , wherein a polymerase with slight or no strand displacement activity is used in Step (c).

7. The method of claim 3 , wherein a polymerase with slight or no strand displacement activity is used in Step (c).

8. The method of claim 1 , wherein the method for random insertion of a tag sequence into a nucleic acid functions for the synthesis of RNA.

9. The method of claim 1 , wherein the method for inserting random insertion of a tag sequence into a nucleic acid serves for the synthesis of DNA.

10. The method of claim 1 , wherein the method for random insertion of a tag sequence into a nucleic acid is used for detecting template nucleic acids.

11. The method of claim 1 , wherein the method for random insertion of a tag sequence into a nucleic acid is used for fusing DNA fragments.

12. The method of claim 1 , wherein methylated DNA sections are selectively amplified.

13. The method of claim 1 , wherein non-methylated DNA sections are selectively amplified.

14. The method of claim 1 , wherein the tag sequence is inserted without subsequent synthesis of the opposite strand at the 3′ end of the newly synthesized nucleic acid.

15. The method of claim 1 , further comprising detecting the new strand of nucleic acid produced in step (c).

16. The method of claim 1 , further comprising amplifying the new strand of nucleic acid produced in step (c).

17. The method of claim 1 , wherein the anchor sequence of the anchor oligonucleotide is a primer.

18. The method of claim 3 , wherein the at least one functional sequence is a hybridization site, a primer binding site, a probe binding site, a promoter, a signal sequence for initiating transcription and/or translation, a restriction endonuclease recognition and restriction site, a ribosome binding site, a protein binding site, an antibody recognition site, or a sequence complementary thereto.

19. The method of claim 18 , wherein the promoter is an RNA polymerase promoter.

20. The method of claim 19 , wherein an in vitro transcription reaction is subsequently performed.

21. The method of claim 1 , wherein the new strand of nucleic acid is processed by means of a sequence-specific endonuclease.

22. The method of claim 21 , wherein the sequence-specific endonuclease is a methylation-sensitive endonuclease.

23. The method of claim 1 , wherein more than one primer is used in step (a), wherein the one or more primers comprises a tag sequence.

24. The method of claim 1 , wherein a double-stranded region of nucleic acid is formed between the new strand of nucleic acid and the non-hybridized portion of the anchor oligonucleotide, wherein the double-stranded region does not exist prior to the formation of the new strand.

25. The method of claim 24 , wherein the double-stranded region of nucleic acid is further processed in a subsequent step (d).

26. The method of claim 25 , wherein the anchor sequence of the anchor oligonucleotide is located at the 3′ region and at least one template tag sequence of at least one anchor oligonucleotide is located in the 5′ region of at least the anchor oligonucleotide.

27. The method of claim 25 , characterized in that the template tag sequence contains at least one functional sequence.

28. The method of claim 25 , wherein a polymerase with slight or no strand displacement activity is used in Step (c).

29. The method of claim 24 , wherein the double-stranded region of nucleic acid is formed by means of a template switch.

30. The method of claim 23 , wherein the sequence of the tag sequence of the primer and the sequence of the template tag sequence of the anchor oligonucleotide are identical.

31. The method of claim 23 , wherein the sequence of the tag sequence of the primer and the sequence of the template tag sequence of the anchor oligonucleotide are different.

32. The method of claim 27 , wherein the at least one functional sequence is a hybridization site, a primer binding site, a probe binding site, a promoter, a signal sequence for initiating transcription and/or translation, a restriction endonuclease recognition and restriction site, a ribosome binding site, a protein binding site, an antibody recognition site, or a sequence complementary thereto.

33. The method of claim 32 , wherein the promoter is an RNA polymerase promoter.

34. The method of claim 33 , wherein an in vitro transcription reaction is subsequently performed.

35. The method of claim 25 , wherein the further processing of step (d) comprises:
(i) ligating the ends of double-stranded nucleic acid strand synthesized in step (c) without prior strand separation, and subsequently
(ii) separating the double-stranded nucleic acid, thereby producing circular single-stranded nucleic acids.

36. The method of claim 32 , wherein the circular single-stranded nucleic acids are amplified by rolling circle amplification.

37. The method of claim 25 , wherein the further processing of step (d) comprises:
(i) separating the new nucleic acid strand from the template strand, and
(ii) forming a hairpin structure through self-hybridization of the ends of the newly synthesized nucleic acid strand.

38. The method of claim 37 , wherein two hairpin structures are ligated to one another to generate a circular nucleic acid.

39. The method of claim 38 , wherein the circular nucleic acid is amplified by rolling circle amplification.

Assignees

• Korfhage Christian
• Engel Holger
• Löffert Dirk
• Peist Ralf
• Qiagen Gmbh

Inventors

• Korfhage Christian
• Engel Holger
• Löffert Dirk
• Peist Ralf

Classifications

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2521/101

  DNA polymerase · CPC title