METHODS AND COMPOSITIONS FOR MULTIPLE DISPLACEMENT AMPLIFICATION OF NUCLEIC ACIDS

• US 20100240102A1
• Filed: 05/30/2008
• Published: 09/23/2010
• Est. Priority Date: 06/01/2007
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Duncan, David A., Eshoo, Mark W.
• Granted Patent: US 9,598,724 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

Disclosed are methods for multiple displacement amplification of a nucleic acid sequence in a sample. The nucleic acid is contacted with a reaction mixture that includes a set of oligonucleotide primers and a plurality of polymerase enzymes. The reaction mixture is subjected to conditions under which the nucleic acid sequence is amplified to produce an amplification product in a multiple displacement amplification reaction. Also disclosed are kits containing a set of oligonucleotide primers with random sequences having lengths of 6 to 8 nucleobases. At least some of the individual members of the primers have one or more ribose modifications that stabilize or lock the ribose ring in a 3′ -endo conformation. At least some of the primers have one or more universal nucleobases.

Claims

1. A method for multiple displacement amplification of a nucleic acid sequence in a sample comprising the steps of:

contacting said nucleic acid with a reaction mixture, wherein said reaction mixture includes a set of oligonucleotide primers and a plurality of polymerase enzymes; and

subjecting said reaction mixture to conditions under which said nucleic acid sequence is amplified to produce an amplified product in a multiple displacement reaction.

2. The method of claim 1 wherein said sample is a forensic sample comprising human DNA.

3. The method of claim 1 wherein said plurality of polymerase enzymes have 5′

→

3′

DNA polymerase activity, 3′

→

5′

exonuclease activity, and 5′

→

3′

excision repair activity.

4. The method of claim 1 wherein members of said set of oligonucleotide primers have random sequences.

5. The method of claim 4 wherein said oligonucleotide primers have lengths of 6, 7 or 8 nucleotide residues.

6. The method of claim 4 wherein said oligonucleotide primers include at least one universal nucleobase.

7. The method of claim 6 wherein said universal nucleobase is inosine.

8. The method of claim 6 or claim 7 wherein said universal nucleobase is located at the 3′

-terminal end of said oligonucleotide primers.

9. The method of claim 1 wherein said set of oligonucleotide primers includes primers having one or more modifications of the ribose ring that favor a 3′

-endo conformation of said ribose ring or lock said ribose ring in said 3′

-endo conformation.

10. The method of claim 9 wherein said modifications comprise a 2′

to 4′

ribose bridge.

11. The method of claim 10 wherein said modifications are located at the 2^nd and 5^th positions of said primers.

12. The method of claim 1 wherein said one or more enzymes include phi29 polymerase and pol I polymerase.

13. The method of claim 1 wherein said one or more enzymes further includes pyrophosphatase.

14. The method of claim 1 including the proviso that bovine serum albumin is not included in said reaction mixture.

15. The method of claim 1 wherein said reaction mixture comprises betaine and trehalose.

16. The method of claim 1 wherein said conditions comprise thermal cycling of said reaction mixture.

17. The method of claim 1 wherein said multiple displacement amplification results in whole genome amplification.

18. The method of claim 1 wherein said multiple displacement amplification reaction produces said amplification product at a constant ratio relative to production of other amplification products of other nucleic acids present in said sample.

19. The method of claim 1 wherein the total quantity of said nucleic acid sequence is between about 2 picograms to about 1000 picograms.

31. The reaction mixture of claim 16 wherein said one or more enzymes further have pyrophosphatase activity.

33. The reaction mixture of claim 18 wherein said compatible solutes comprise betaine.

34. The reaction mixture of claim 18 wherein said compatible solutes comprise trehalose.

35. The reaction mixture of claim 18 wherein said compatible solutes comprise betaine and trehalose.

36. The reaction mixture of claim 18 with the proviso that bovine serum albumin is not included in said reaction mixture.

38. The kit of claim 35 wherein said individual primers further comprise one or more phosphorothioate linkages.

39. The kit of claim 35 wherein said set of oligonucleotide primers include at least some heptamer primers.

40. The kit of claim 38 wherein said universal nucleobase is inosine.

41. The kit of claim 39 wherein said heptamers have inosine residues at the 6^th and 7^th positions.

42. The kit of claim 40 wherein said heptamers have one or more phosphorothioate linkages.

43. The kit of claim 41 wherein said phosphorothioate linkages are located between the 5^th and 6^th residue and between the 6^th and 7^th residue of said heptamers.

44. The kit of claim 35 wherein said ribose modifications stabilize a 3′

-endo conformation of said ribose ring or lock said ribose ring in said 3′

-endo conformation.

45. The kit of claim 43 wherein said ribose modifications include a 2′

to 4′

ribose bridge.

46. The kit of claim 43 or claim 44 wherein said modifications are located at the 2^nd and 5^th positions of said primers.

47. The kit of claim 33 further comprising a plurality of enzymes having 5′

→

3′

DNA polymerase activity, 3′

→

5′

exonuclease proofreading activity, and 5′

→

3′

nick translation DNA repair activity.

48. The kit of claim 46 wherein said one or more enzymes further have pyrophosphatase activity.

49. The kit of claim 46 wherein said plurality of enzymes comprise Phi29 polymerase, DNA polymerase I, and pyrophosphatase.

50. The kit of claim 35 further comprising one or more compatible solutes.

51. The kit of claim 49 wherein said compatible solutes comprise betaine.

52. The kit of claim 49 wherein said compatible solutes comprise trehalose.

53. The kit of claim 49 wherein said compatible solutes comprise betaine and trehalose.

54. The kit of claim 35 further comprising a set of natural deoxynucleotide triphosphates.

55. The kit of claim 35 further comprising instructions for performing said multiple displacement amplification of said nucleic acid sample.

57. The oligonucleotide primer of claim 55 wherein said universal base is inosine.

59. A kit comprising a plurality of primers according to claim 55.

60. The kit of claim 57 further comprising instructions for performing a multiple displacement amplification reaction on a nucleic acid sample.

20. A reaction mixture for multiple displacement amplification of a nucleic acid sample comprising:

a set of oligonucleotide primers;

a plurality of polymerase enzymes;

a set of natural deoxynucleotide triphosphates; and

one or more compatible solutes.

21. The reaction mixture of claim 20 wherein said set of oligonucleotide primers comprises primers having lengths of 6 to 8 nucleobases.

22. The reaction mixture of claim 20 wherein said set of oligonucleotide primers comprises random sequence primers.

23. The reaction mixture of claim 20 wherein said set of oligonucleotide primers comprises primers having at least one universal nucleobase.

24. The reaction mixture of claim 23 wherein said universal nucleobase is inosine.

25. The reaction mixture of claim 23 wherein said set of oligonucleotide primers comprises heptamers having inosine residues at the 6^th and 7^th positions.

26. The reaction mixture of claim 25 wherein said heptamers have one or more phosphorothioate linkages.

27. The reaction mixture of claim 26 wherein said phosphorothioate linkages are located between the 5^th and 6^th residue and between the 6^th and 7^th residue of said heptamers.

28. The reaction mixture of claim 20 wherein said set of oligonucleotide primers includes primers having one or more nucleotide residues with a ribose modification that stabilizes a 3′

-endo conformation of said ribose ring or locks said ribose ring in said 3′

-endo conformation.

29. The reaction mixture of claim 28 wherein said ribose modification is bicyclic nucleic acid.

30. The reaction mixture of claim 28 or claim 29 wherein said nucleotide residues are located at the 2^nd and 5^th positions of said primers.

32. The reaction mixture of claim 27 wherein said enzymes comprise Phi29 polymerase, DNA polymerase I, and pyrophosphatase.

37. A kit for multiple displacement amplification of a nucleic acid sample comprising:

a set of oligonucleotide primers with random sequences having lengths of 6 to 8 nucleobases wherein at least some individual primers of said set of oligonucleotide primers comprise;

one or more ribose modifications that stabilize a 3′

-endo conformation of said ribose ring or lock said ribose ring in said 3′

-endo conformation; and

one or more universal nucleobases.

56. An oligonucleotide primer comprising:

a heptamer oligonucleotide having;

a random nucleobase sequence from the first to the fifth positions of said heptamer;

a universal nucleobase at the sixth and seventh positions of said heptamer; and

a ribose modification at the second and fifth positions, said modification comprising a 2′

to 4′

ribose bridge.

58. The oligonucleotide primer of claim 56 further comprising one or more phosphorothioate linkages at the 3′

end.