Kits including stem-loop oligonucleotides for use in preparing nucleic acid molecules

• US 10,196,686 B2
• Filed: 02/13/2017
• Issued: 02/05/2019
• Est. Priority Date: 08/02/2005
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Makarov, Vladimir L., Kamberov, Emmanuel, Tarrier, Brendan J.
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/430,803
• Publication Number:
  US 20170314069A1
• Time in Patent Office: 722 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Claims

1. A kit comprising:

a stem-loop oligonucleotide comprising;

an inverted repeat;

a loop;

a 5′

end that lacks a phosphate; and

a non-replicable base or sequence, or at least one deoxy-uridine.

2. The kit according to claim 1, wherein the stem loop oligonucleotide comprises a non-replicable base or sequence.

3. The kit according to claim 2, wherein at least part of the non-replicable base or sequence is present in the loop of the oligonucleotide or in a sequence of the stem adjacent to the loop.

4. The kit according to claim 1, wherein the non-replicable base or sequence comprises an abasic site or sequence, hexaethylene glycol, or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.

5. The kit according to claim 1, wherein the stem loop oligonucleotide comprises at least one deoxy-uridine.

6. The kit according to claim 5, wherein the loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.

7. The kit according to claim 6, wherein the kit further comprises deoxy-uridine glycosylase.

8. The kit according to claim 1, wherein the kit further comprises a ligase.

9. The kit according to claim 1, wherein the kit further comprises a DNA polymerase.

10. The kit according to claim 1, wherein the 5′

end that lacks a phosphate comprises a 5′

OH.

11. The kit according to claim 1, wherein the kit further comprises an endonuclease.

12. The kit according to claim 1, wherein the kit further comprises a thermostable polymerase.

13. The kit according to claim 12, wherein the kit further comprises a universal primer.

14. The kit according to claim 12, wherein the kit further comprises dNTPs.

15. The kit according to claim 5, wherein the kit further comprises a ligase.

16. The kit according to claim 15, wherein the kit further comprises deoxy-uridine glycosylase.

17. The kit according to claim 15, wherein the kit further comprises a DNA polymerase.

18. The kit according to claim 15, wherein the kit further comprises an endonuclease.

19. The kit according to claim 15, wherein the kit further comprises a thermostable polymerase and dNTPs.

20. The kit according to claim 19, wherein the kit further comprises a universal primer.

21. The kit according to claim 8, wherein the ligase and the stem-loop oligonucleotide are present in a single solution.

22. The kit according to claim 7, wherein the kit further comprises a thermostable polymerase in a single solution with the deoxy-uridine glycosylase.

23. The kit according to claim 9, wherein the DNA polymerase has end-polishing activity.

24. The kit according to claim 17, wherein the DNA polymerase has end-polishing activity.