Insertion of Sequence Elements into Nucleic Acids

• US 20080057543A1
• Filed: 05/04/2007
• Published: 03/06/2008
• Est. Priority Date: 05/05/2006
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Peist, Ralf, Korfhage, Christian, Engel, Holger, Loffert, Dirk
• Granted Patent: US 8,932,831 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/10 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 Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

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.

Claims

1. A method for inserting one or more tag sequences into a nucleic acid comprising the following steps:

(a) providing a template nucleic acid;

(b) hybridizing at least one anchor sequence of at least one anchor oligonucleotide with at least one sequence section of the template nucleic acid, wherein the anchor oligonucleotide comprises a template tag sequence;

(c) synthesizing a new strand of nucleic acid, wherein the new strand of nucleic acid is partially complementary to the template nucleic acid, wherein the new strand of nucleic acid contains a sequence complementary to a non-hybridized portion of the anchor oligonucleotide, wherein a double-stranded region of nucleic acid is formed.

2. The method of claim 1, wherein the sequence complementary to the non-hybridized portion of the anchor oligonucleotide is complementary to at least one template tag sequence on the 3′

end of the non-hybridized portion of the anchor oligonucleotide.

3. The method of claim 1, wherein the double-stranded region of nucleic acid produced in Step (c) is further processed in a subsequent step (d).

4. The method of claim 1, wherein at least one anchor sequence of at least one 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 one anchor oligonucleotide.

5. The method of claim 3, wherein at least one anchor sequence of at least one 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 one anchor oligonucleotide.

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

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

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

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

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

11. The method of claim 4, wherein a polymerase with slight or no strand displacement activity is used in Step (c).

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

13. The method of claim 1, wherein Step (c) is performed with a primer, from which the synthesis of the new strand of nucleic acid begins.

14. The method of claim 1, wherein the method for inserting a tag sequence into a nucleic acid functions for the synthesis of RNA.

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

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

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

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

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

20. The use of an anchor oligonucleotide in a method according to claim 1.

21. A nucleic acid, which is synthesized in a first-strand synthesis, characterized in that it has a tag sequence at its 3′

end, inserted during first-strand synthesis in a template-dependent manner, whereby the sequence complementary to the tag sequence is not a part of the template nucleic acid.

22. The nucleic acid according to claim 21, wherein the tag sequence is inserted at the 3′

end by means of an anchor oligonucleotide.

23. An anchor oligonucleotide having an anchor sequence in the 3′

region and a template tag sequence in the 5′

region, whereby the anchor sequence is hybridized with a template nucleic acid during the synthesis of the new strand of nucleic acid and the template tag sequence is not hybridized with the template nucleic acid, characterized in that the synthesis of a new strand of nucleic acid, which is complementary to a part of the template nucleic acid and whose synthesis begins 3′

of the hybridizing site of the anchor sequence of the anchor oligonucleotide at the template strand, ends by means of a template switch of the polymerase at the 5′

end of the template tag sequence of the anchor oligonucleotide.