COMPOSITIONS AND METHODS FOR PROCESSING AND AMPLIFICATION OF DNA, INCLUDING USING MULTIPLE ENZYMES IN A SINGLE REACTION

• US 20110081685A1
• Filed: 09/28/2010
• Published: 04/07/2011
• 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.
• Granted Patent: US 8,071,312 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• 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; and

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.

21. 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 and wherein polymerization of an oligonucleotide-ligated DNA fragment excludes at least part of the inverted repeat by subjecting the oligonucleotide-ligated 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.

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

23. The method of claim 22, 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.

24. The method of claim 22, 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.

25. The method of claim 24, wherein the abasic site or sequence is introduced by one or more enzymes in the single solution.

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

28. 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 an oligonucleotide-ligated DNA fragment, and wherein strand displacement or nick translation polymerization of the oligonucleotide-ligated DNA fragment generates an endonuclease site.

37. 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.

38. The method of claim 37, wherein the recognition sequence for a nicking endonuclease resides at least in part within the stem of the stem-loop oligonucleotide.

39. The method of claim 37, wherein the recognition sequence for a nicking endonuclease resides at least in part within the loop of the stem-loop oligonucleotide.

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

end of the stem-loop oligonucleotide lacks a phosphate.

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

42. The method of claim 41, wherein the further modification comprises cloning.

43. The method of claim 42, further defined as comprising incorporation of the modified molecule into a vector, said incorporation occurring at ends in the modified molecule generated by endonuclease cleavage within the inverted repeat.

44. The method of claim 1, further defined as occurring in a single suitable solution, wherein the process occurs in the absence of exogenous manipulation.

45. The method of claim 44, further defined as occurring at one temperature.

46. The method of claim 44, 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.

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

49. The method of claim 48, wherein the molecule is immobilized non-covalently.

50. The method of claim 48, wherein the molecule is immobilized covalently.

51. The method of claim 1, further comprising the step of digesting the DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes attached to one strand of the DNA fragment, wherein 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 generates a 5′

overhang, and wherein the single stranded 5′

overhang hybridizes to a complementary oligonucleotide covalently immobilized to a solid support.

2-20. -20. (canceled)

52-54. -54. (canceled)