Methods of performing nucleic acid amplification assays using modified primers

• US 8,048,633 B2
• Filed: 06/29/2010
• Issued: 11/01/2011
• Est. Priority Date: 12/23/2004
• Status: Expired due to Fees

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Inventors: Miller, Cary James, Collier, Gordon Bruce, MacLeod, Jason Andrew, Dicke, William Charles, Wood, John Allister, Nemeth, Attila Csaba
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US12/826,450
• Publication Number:
  US 20100330575A1
• Time in Patent Office: 490 Days
• Field of Search: 435/6, 435/91.2, 435/6.12, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502769 characterised by multiphase flow arrangements
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/0335 using coils
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical or biological applications
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]

Abstract

The invention relates to a method for the extraction of nucleic acid from cells, the amplification of segments of nucleic acid using modified primers, and the detection of nucleic acids.

Claims

1. A method of performing a nucleic acid amplification assay comprising:

(a) combining reagents, DNA polymerase, a target nucleic acid, and a modified primer,

said modified primer comprising a sequence of bases complementary to a first region of said target nucleic acid, a polymerase blocking region, a single stranded hybridization region attached to said polymerase blocking region, and a detectable label, said primer predominantly having a secondary structure in solution in a lower temperature range and not in an elevated temperature range, wherein said secondary structure involves annealing one or more self-complementary regions between portions of said primer region and portions of said single stranded hybridization region, wherein said modified primer is capable of priming said target nucleic acid and hybridizing with an oligonucleotide complementary to said single stranded hybridization region at a first elevated temperature range, and substantially not in a second lower temperature range;

(b) cycling the mixture of (a) at said first temperature range to provide multiple copies of an amplicon incorporating said modified primer, wherein said incorporated primer is capable of hybridizing with an oligonucleotide complementary to said single stranded hybridization region in said first elevated temperature range and in said second lower temperature range;

(c) reducing the temperature of the mixture to the second temperature range;

(d) exposing said mixture to a capture oligonucleotide complementary to said single stranded hybridization region;

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer, with said capture oligonucleotide; and

(f) detecting said label associated with said hybridization.

2. The method of claim 1, wherein said target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and modifications and derivatives thereof.

3. The method of claim 1, wherein said target nucleic acid is extracted from blood, a buccal swab, tissue, a bodily fluid, an environmental sample, a surface of a material, a plant, an animal, a bacteria or a fungi.

4. The method of claim 1, wherein said reagents comprise reagents for a polymerase chain reaction amplification.

5. The method of claim 1, wherein said DNA polymerase is Taq polymerase.

6. The method of claim 1, wherein said DNA polymerase is thermococcus kodakiensis polymerase.

7. The method of claim 1, wherein said primer is selected from the group deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid and hexa-polyethylene glycol modified nucleic acid.

8. The method of claim 1, wherein said polymerase blocking region is a phosphoramidite.

9. The method of claim 1, wherein said polymerase blocking region comprises 18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

10. The method of claim 1, wherein said single stranded hybridization region is selected from the group consisting of nucleic acids, ribonucleic acids, peptide nucleic acids, and modified nucleic acids.

11. The method of claim 1, wherein said sequence of bases complementary to a first region has a reactive 3′

-hydroxyl.

12. The method of claim 1, wherein said detectable label is selected from the group consisting of biotin, streptavidin, FAM, DNP, cholesterol, and a DNA glycoconjugate.

13. The method of claim 1, wherein said detectable label is biotin.

14. The method of claim 1, wherein cycling is isothermal.

15. The method of claim 1, wherein cycling is between a first and second temperature.

16. The method of claim 1, wherein said capture oligonucleotide is immobilized on an optical surface.

17. The method of claim 1, wherein said capture oligonucleotide is immobilized on a bead.

18. The method of claim 1, wherein said capture oligonucleotide is immobilized on an electrode.

19. The method of claim 1, wherein the single stranded hybridization region of the modified primer hybridizes to the capture oligonucleotide.

20. The method of claim 1, wherein said detection is electrochemical.

21. The method of claim 1, wherein said detection is an optical method.

22. The method of claim 1, wherein said detection is colorimetric.

23. The method of claim 1, wherein said detection is fluorescent.

24. The method of claim 1, wherein said detection is enzymatic.

25. The method of claim 1, wherein said label is exposed to a second moiety which has a second label, said second moiety binding specifically to the first label, and wherein detection involves detection of said second label.

26. The method of claim 1, wherein said label is exposed to a second moiety which has a second label, said second moiety binding specifically to said first label, wherein detection involves detection of said second label, wherein said label is selected from the group alkaline phosphatase, glucose oxidase, sarcosine oxidase, cholesterol oxidase, uricase, L-amino acid oxidase, D-amino acid oxidase, urease, ascorbate oxidase, horseradish peroxidase and luciferase.

27. The method of claim 1, wherein said label is biotin and is exposed to a streptavidin-labeled alkaline phosphatase and detection of said label is based on detection of alkaline phosphatase activity.

28. The method of claim 1, wherein said label is biotin and is exposed to a streptavidin-labeled alkaline phosphatase and detection of biotin is based on detection of said alkaline phosphatase, and wherein alkaline phosphatase converts p-aminophenol phosphate to p-aminophenol and is detected electrochemically.

29. A method of performing a nucleic acid amplification assay comprising:

(a) combining reagents, DNA polymerase, a target nucleic acid, a modified primer and a second primer,

said modified primer comprising a sequence of bases complementary to a first region of said target nucleic acid, a polymerase blocking region and a single stranded hybridization region attached to said polymerase blocking region, said primer predominantly having a secondary structure in solution in a lower temperature range and not in an elevated temperature range, wherein said secondary structure involves annealing one or more self-complementary regions between portions of said primer region and portions of said single stranded hybridization region,

wherein said modified primer is capable of priming said target nucleic acid and hybridizing with an oligonucleotide complementary to said single stranded hybridization region at a first elevated temperature range, and substantially not in a second lower temperature range,

said second primer comprising a sequence of bases complementary to a second region of said target nucleic acid and having a detectable label;

(b) cycling the mixture of (a) at said first temperature range to provide multiple copies of an amplicon incorporating said modified primer and said second primer, wherein said incorporated modified primer is capable of hybridizing with an oligonucleotide complementary to said single stranded hybridization region in said first elevated temperature range and in said second lower temperature range;

(c) reducing the temperature of the mixture to the second temperature range;

(d) exposing said mixture to a capture oligonucleotide complementary to said single stranded hybridization region;

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer, with said capture oligonucleotide; and

(f) detecting said label associated with said second primer incorporated into said amplicon.

30. The method of claim 29, wherein said target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and modifications and derivatives thereof.

31. The method of claim 29, wherein said target nucleic acid is extracted from blood, a buccal swab, tissue, a bodily fluid, an environmental sample, a surface of a material, a plant, an animal, a bacteria or a fungi.

32. The method of claim 29, wherein said reagents comprise reagents for a polymerase chain reaction amplification.

33. The method of claim 29, wherein said DNA polymerase is Taq polymerase.

34. The method of claim 29, wherein said DNA polymerase is thermococcus kodakiensis polymerase.

35. The method of claim 29, wherein said primer is selected from the group deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid and hexa-polyethylene glycol modified nucleic acid.

36. The method of claim 29, wherein said polymerase blocking region is a phosphoramidite.

37. The method of claim 29, wherein said polymerase blocking region comprises 18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

38. The method of claim 29, wherein said single stranded hybridization region is selected from the group consisting of nucleic acids, ribonucleic acids, peptide nucleic acids, and modified nucleic acids.

39. The method of claim 29, wherein said sequence of bases complementary to a first region has a reactive 3′

-hydroxyl.

40. The method of claim 29, wherein said detectable label is selected from the group consisting of biotin, streptavidin, FAM, DNP, cholesterol, and a DNA glycoconjugate.

41. The method of claim 29, wherein said detectable label is biotin.

42. The method of claim 29, wherein cycling is isothermal.

43. The method of claim 29, wherein cycling is between a first and second temperature.

44. The method of claim 29, wherein said capture oligonucleotide is immobilized on an optical surface.

45. The method of claim 29, wherein said capture oligonucleotide is immobilized on a bead.

46. The method of claim 29, wherein said capture oligonucleotide is immobilized on an electrode.

47. The method of claim 29, wherein the single stranded hybridization region of the modified primer hybridizes to the capture oligonucleotide.

48. The method of claim 29, wherein said detection is electrochemical.

49. The method of claim 29, wherein said detection is an optical method.

50. The method of claim 29, wherein said detection is colorimetric.

51. The method of claim 29, wherein said detection is fluorescent.

52. The method of claim 29, wherein said detection is enzymatic.

53. The method of claim 29, wherein said label is exposed to a second moiety which has a second label, said second moiety binding specifically to the first label, and wherein detection involves detection of said second label.

54. The method of claim 29, wherein said label is exposed to a second moiety which has a second label, said second moiety binding specifically to said first label, wherein detection involves detection of said second label, wherein said label is selected from the group alkaline phosphatase, glucose oxidase, sarcosine oxidase, cholesterol oxidase, uricase, L-amino acid oxidase, D-amino acid oxidase, urease, ascorbate oxidase, horseradish peroxidase and luciferase.

55. The method of claim 29, wherein said label is biotin and is exposed to a streptavidin-labeled alkaline phosphatase and detection of said label is based on detection of alkaline phosphatase activity.

56. The method of claim 29, wherein said label is biotin and is exposed to a streptavidin-labeled alkaline phosphatase and detection of biotin is based on detection of said alkaline phosphatase, and wherein alkaline phosphatase converts p-aminophenol phosphate to p-aminophenol and is detected electrochemically.