Methods for producing and using stem-loop oligonucleotides

• US 8,071,312 B2
• Filed: 09/28/2010
• Issued: 12/06/2011
• Est. Priority Date: 08/02/2005
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Makarov, Vladimir L., Kamberov, Emmanuel, Tarrier, Brendan J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US12/892,359
• Publication Number:
  US 20110081685A1
• Time in Patent Office: 434 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.12
• 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 method of preparing a nucleic acid molecule, comprising:

providing a double stranded nucleic acid molecule;

attaching one strand of a stem-loop oligonucleotide comprising an inverted repeat and a loop to the double stranded nucleic acid molecule to produce an oligonucleotide-attached nucleic acid molecule; and

i) subjecting the oliognucleotide-attached nucleic acid molecule to strand displacement or nick translation polymerization to generate an endonuclease site or

ii) wherein the stem-loop oligonucleotide further comprises a recognition sequence for a nicking endonuclease and wherein the oligonucleotide-attached nucleic acid molecule is amplified by strand displacement synthesis and second strand synthesis.

2. The method of claim 1, further comprising the steps of digesting the double stranded DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes ligated to one strand of the DNA fragment and subjecting the oligonucleotide-attached DNA fragment to strand displacement or nick translation polymerization that halts at a base or sequence in the loop or in a region of the stem adjacent to the loop to thereby exclude at least part of the inverted repeat.

3. The method of claim 1, wherein the stem-loop oligonucleotide is further defined as comprising a non-replicable base or sequence.

4. The method of claim 3, 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.

5. The method of claim 3, 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.

6. The method of claim 5, wherein the non-replicable base or sequence comprises an abasic site or sequence.

7. The method of claim 1, wherein the loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.

8. The method of claim 1, comprising subjecting the oligonucleotide-attached nucleic acid to strand displacement or nick translation polymerization to generate an endonuclease site.

9. The method of claim 1, further comprising the step of digesting the double stranded DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes ligated to one strand of the DNA fragment to produce the oligonucleotide-ligated DNA fragment.

10. The method of claim 8, wherein the endonuclease site is a site-specific restriction endonuclease site and wherein at least part of the inverted repeat is removed by cleavage with said restriction endonuclease.

11. The method of claim 8, wherein the endonuclease site resides within the stem and loop regions.

12. The method of claim 8, wherein the endonuclease site resides within the loop region.

13. The method of claim 8, wherein the endonuclease site resides within the stem region.

14. The method of claim 8, wherein the endonuclease site is a homing endonuclease site.

15. The method of claim 1, further comprising initiating RNA transcription from a regulatory sequence positioned on said nucleic acid molecule, thereby producing at least one transcribed polynucleotide.

16. The method of claim 15, wherein the transcribed polynucleotide is replicated by a reverse transcriptase.

17. The method of claim 16, wherein reverse transcription is initiated by hybridization of an oligonucleotide complementary to the 3′

end of the transcribed polynucleotide.

18. The method of claim 1, wherein the stem-loop oligonucleotide further comprises a recognition sequence for a nicking endonuclease and wherein the oligonucleotide-attached nucleic acid molecule is amplified by strand displacement synthesis and second strand synthesis.

19. The method of claim 18, wherein the recognition sequence for a nicking endonuclease resides at least in part within the stem of the stem-loop oligonucleotide.

20. The method of claim 18, wherein the recognition sequence for a nicking endonuclease resides at least in part within the loop of the stem-loop oligonucleotide.

21. The method of claim 1, wherein a 5′

end of the stem-loop oligonucleotide lacks a phosphate.

22. The method of claim 1, wherein the oligonucleotide-attached nucleic acid molecule is further modified.

23. The method of claim 22, further comprising incorporating the modified molecule into a vector, said incorporation occurring at ends in the modified molecule generated by endonuclease cleavage within the inverted repeat.

24. The method of claim 1, further defined as occurring in a single suitable solution.

25. The method of claim 24, further defined as occurring at one temperature.

26. The method of claim 24, wherein the solution comprises one or more of the following:

ligase;

DNA polymerase;

one or more endonucleases;

RNA polymerase;

reverse transcriptase;

RNase H;

deoxy-uridine glycosylase;

nickase;

thermophilic DNA polymerase;

ATP;

rNTPs; and

dNTPs.

27. The method of claim 1, wherein the oligonucleotide-attached nucleic acid molecule is immobilized to a solid support.

28. The method of claim 27, wherein the oligonucleotide-attached nucleic acid molecule is immobilized non-covalently.

29. The method of claim 27, wherein the oligonucleotide-attached nucleic acid molecule is immobilized covalently.

30. The method of claim 1, further comprising the steps of digesting the DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes attached to one strand of the DNA fragment, and generating a single-stranded 5′

overhang by strand displacement polymerization of the oligonucleotide-ligated DNA fragment and its arrest at a base or sequence in the loop or in a region of the stem adjacent to the loop.