Methods for processing and amplifying nucleic acids

• US 9,598,727 B2
• Filed: 04/11/2014
• Issued: 03/21/2017
• 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
• Inventors: Makarov, Vladimir L., Kamberov, Emmanuel, Tarrier, Brendan J.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US14/250,538
• Publication Number:
  US 20150079637A1
• Time in Patent Office: 1,075 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 1/1
• 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 is directed generally to the use of stem-loop oligonucleotides comprising an inverted repeat, a loop, and at least one degradable site or site capable of becoming a degradable site in the preparation of DNA molecules, such as a library. 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 producing a primer for use in an amplification reaction comprising:

(a) providing at least one stem-loop oligonucleotide comprising an inverted repeat, a loop, and at least one degradable site or site capable of becoming a degradable site;

(b) subjecting the at least one stem-loop oligonucleotide to conditions such that a phosphodiester bond of the at least one stem-loop oligonucleotide at the at least one degradable site or site capable of becoming a degradable site is broken, thereby producing primers for use in an amplification reaction; and

(c) carrying out an amplification reaction using the primers.

2. The method of claim 1, wherein the conditions of step (b) comprise heating the at least one stem-loop oligonucleotide.

3. The method of claim 2, wherein the conditions of step (b) comprises incubating the stem-loop oligonucleotide with uracil-DNA glycosylase to convert the at least one site capable of becoming a degradable site into an abasic site prior to said heating.

4. The method of claim 1, wherein prior to step (b) a portion of the at least one stem-loop oligonucleotide is ligated to a nucleic acid molecule to provide a stem-loop oligonucleotide ligated nucleic acid molecule, and the amplification step is carried out on the stem-loop ligated nucleic acid molecule.

5. The method of claim 4, wherein the nucleic acid molecule is a double stranded DNA molecule.

6. The method of claim 4, wherein the nucleic acid molecule is a double stranded DNA/RNA hybrid.

7. The method of claim 1, wherein the amplification is a polymerase chain reaction.

8. The method of claim 1, wherein the amplification is a linear amplification.

9. The method of claim 1, wherein the amplification reaction is carried out on a nucleic acid molecule, such that the nucleic acid molecule is amplified.

10. The method of claim 9, further defined as occurring in a single suitable solution, wherein the method occurs in the absence of exogenous manipulation.

11. The method of claim 10, wherein the solution comprises one or more of the following:

an adaptor molecule;

ligase;

DNA polymerase;

one or more endonucleases;

RNA polymerase;

reverse transcriptase;

RNase H;

uracil-DNA glycosylase;

nickase;

thermophilic DNA polymerase;

ATP;

rNTPs; and

dNTPs.

12. The method of claim 1, wherein the degradable site is an abasic site.

13. The method of claim 12, wherein the conditions sufficient to break the phosphodiester bonds of the degradable site comprise exposure to heat.

14. The method of claim 1, wherein the site capable of becoming a degradable site is further defined as a base that can be enzymatically converted to a degradable site.

15. The method of claim 14, wherein the base that can be enzymatically converted to a degradable site is deoxyuridine.

16. The method of claim 15, wherein the conditions sufficient to break the phosphodiester bonds of the site capable of becoming a degradable site comprise incubating the stem-loop oligonucleotide with uracil-DNA glycosylase to convert the deoxyuridine into an abasic.

17. The method of claim 16, wherein the conditions further comprise exposing the stem-loop oligonucleotide to heat.

18. The method of claim 16, wherein the conditions further comprise incubating the stem-loop oligonucleotide with endonuclease VIII.

19. The method of claim 1, wherein the stem-loop oligonucleotide comprises:

(i) a 5′

portion comprising a sequence at least 80% complementary to the 3′

primer portion;

(ii) a loop portion coupled to the 3′

-end of the 5′

portion; and

(iii) a 3′

primer portion coupled to the 3′

-end of the loop portion,

wherein the 5′

portion and/or the loop portion comprise at least one degradable site or site capable of becoming a degradable site.

20. The method of claim 19, wherein the 5′

portion and/or the loop portion comprise 2, 3, 4, 5, or 6 degradable sites or sites capable of becoming degradable sites.

21. The method of claim 1, wherein the loop portion comprises a known sequence.

22. The method of claim 21, wherein the known sequence is a regulatory sequence, an endonuclease recognition sequence, a hybridization sequence, or a primer binding sequence.

23. The method of claim 1, wherein the loop portion comprises a non-replicable base or sequence.

24. The method of claim 23, wherein the non-replicable base or sequence is an abasic site or sequence, hexaethylene glycol, or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.

25. The method of claim 19, wherein the 3′

primer portion is complementary to a known target sequence or sequences.

26. The method of claim 25, wherein the known target sequence is a library adaptor sequence.

27. The method of claim 25, wherein the known target sequence is a genomic sequence or sequences.

28. The method of claim 1, wherein the stem-loop oligonucleotide comprises a blunt end.

29. The method of claim 1, wherein the stem-loop oligonucleotide comprises an overhanging end.

30. The method of claim 1, wherein the 5′

end lacks a phosphate.

31. The method of claim 1, wherein the 3′

end is not ligation-competent.

32. The method of claim 31, wherein the 3′

end is recessed.

33. The method of claim 31, wherein the 3′

end comprises a blocked nucleotide.