Compositions including a double stranded nucleic acid molecule and a stem-loop oligonucleotide

• US 10,208,337 B2
• Filed: 08/10/2017
• Issued: 02/19/2019
• 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:
  US15/674,468
• Publication Number:
  US 20170342475A1
• Time in Patent Office: 558 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. An oligonucleotide-attached nucleic acid molecule comprising:

a double stranded nucleic acid molecule comprising a first strand and a second strand; and

a stem-loop oligonucleotide comprising;

an inverted repeat;

a loop;

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

a 5′

end that lacks a phosphate;

wherein the stem-loop oligonucleotide is attached to the first strand of the double stranded nucleic acid molecule and the 5′

end of the stem-loop oligonucleotide is separated from the second strand of the double stranded nucleic acid molecule.

2. The oligonucleotide-attached nucleic acid molecule according to claim 1, wherein the double stranded nucleic acid molecule is a double stranded DNA molecule.

3. The oligonucleotide-attached nucleic acid molecule according to claim 1, wherein the 5′

end is separated from the second strand of the double stranded nucleic acid molecule by a nick, a gap, or a 5′

flap structure.

4. The oligonucleotide-attached nucleic acid molecule according to claim 1, wherein the 5′

end that lacks a phosphate comprises a 5′

OH.

5. The oligonucleotide-attached nucleic acid molecule according to claim 1, wherein the stem loop oligonucleotide comprises a non-replicable base or sequence.

6. The oligonucleotide-attached nucleic acid molecule according to claim 5, 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.

17. The oligonucleotide-attached nucleic acid molecule according to claim 6, wherein the non-replicable base or sequence comprises an abasic site or sequence, or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.

19. A composition comprising:

the oligonucleotide-attached nucleic acid molecule according to claim 1; and

a ligase, a polymerase, a glycosylase, or a combination thereof.

20. The composition according to claim 19, wherein the polymerase is a thermostable polymerase.

7. An oligonucleotide-attached nucleic acid molecule comprising:

a double stranded nucleic acid molecule and a stem-loop oligonucleotide comprising an inverted repeat and a loop, wherein one strand of the stem-loop oligonucleotide is attached to one strand of the double stranded nucleic acid molecule and the stem loop oligonucleotide further comprises at least one deoxy-uridine.

8. The oligonucleotide-attached nucleic acid molecule according to claim 7, wherein the loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.

9. A composition comprising a plurality of oligonucleotide-attached nucleic acid molecules each comprising:

a double stranded nucleic acid molecule comprising a first strand and a second strand; and

a stem-loop oligonucleotide comprising;

an inverted repeat;

a loop;

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

a 5′

end that lacks a phosphate;

wherein the stem-loop oligonucleotide is attached to the first strand of the double stranded nucleic acid molecule and the 5′

end of the stem-loop oligonucleotide is separated from the second strand of the double stranded nucleic acid molecule.

10. The composition according to claim 9, wherein the double stranded nucleic acid molecule is a double stranded DNA molecule.

11. The composition according to claim 9, wherein the 5′

end is separated from the second strand of the double stranded nucleic acid molecule by a nick, a gap, or a 5′

flap structure.

12. The composition according to claim 9, wherein the 5′

end that lacks a phosphate comprises a 5′

OH.

13. The composition according to claim 9, wherein the stem loop oligonucleotide comprises a non-replicable base or sequence.

14. The composition according to claim 13, 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.

18. The oligonucleotide-attached nucleic acid molecule according to claim 14, wherein the non-replicable base or sequence comprises an abasic site or sequence, or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.

15. A composition comprising a plurality of oligonucleotide-attached nucleic acid molecules each comprising:

a double stranded nucleic acid molecule; and

a stem-loop oligonucleotide comprising an inverted repeat and a loop, wherein one strand of the stem-loop oligonucleotide is attached to one strand of the double stranded nucleic acid molecule and wherein the stem loop oligonucleotide further comprises at least one deoxy-uridine.

16. The composition according to claim 15, wherein the loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.