Materials and kits for use in hot-start PCR, and methods of amplifying nucleic acids in a polymerase chain reaction

• US 20060263811A1
• Filed: 05/03/2006
• Published: 11/23/2006
• Est. Priority Date: 05/03/2005
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Jeon, GeunSook, Vann, Charles S., Lau, Aldrich N. K., Khan, Shaheer H., Karger, Achim E.
• Application Number:
  US11/416,784
• Publication Number:
  US 20060263811A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2549/101 Hot start

Abstract

Materials for sequestering reagents in hot-start PCR, kits containing such materials, and methods for the use of such materials in amplifying nucleic acids are described.

Claims

1. A material for use in hot-start PCR comprising:

a polylactone matrix; and

a PCR reagent;

wherein the polylactone matrix substantially sequesters the PCR reagent from participation in a polymerase chain reaction at ambient temperature.

2. The material of claim 1 wherein the polylactone matrix comprises a polylactone selected from the group consisting of poly(β

-propiolactone), poly(β

-butyrolactone), poly(γ

-butyrolactone), poly(α

-methyl-γ

-butyrolactone), poly(γ

-methyl-γ

-hexanolactone), poly(γ

-valerolactone), poly(γ

-caprolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof.

3. The material of claim 1 wherein the polylactone matrix comprises a polylactone selected from the group consisting of poly(β

-propiolactone), poly(γ

-butyrolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof.

4. The material of claim 1 wherein the polylactone matrix comprises poly(ε

-caprolactone).

5. The material of claim 1 wherein the PCR reagent is selected from the group consisting of oligonucleotides, deoxynucleoside triphosphates, polymerase enzymes, metal ions, and combinations thereof.

6. The material of claim 5 wherein the oligonucleotides are selected from the group consisting of antisense primers, sense primers, and a combination thereof.

7. The material of claim 5 wherein the deoxynucleoside triphosphates are selected from the group consisting of dATP, dTTP, dCTP, dGTP, dUTP, dITP, and combinations thereof.

8. The material of claim 5 wherein the polymerase enzymes comprise a heat-stable DNA polymerase.

9. The material of claim 8 wherein the heat-stable DNA polymerase comprises an enzyme from a species selected from the group consisting of Thermus, Bacillus, Thermococcus, Thermotoga, Pyrococcus, and combinations thereof.

10. The material of claim 9 wherein the Thermus is selected from the group consisting of Thermus aquaticus, Thermus thermophilus, and a combination thereof.

11. The material of claim 9 wherein the Bacillus comprises Bacillus stearothermophilus.

12. The material of claim 9 wherein the Thermococcus comprises Thermococcus litoralis.

13. The material of claim 9 wherein the Thermotoga comprises Thermotoga maritama.

14. The material of claim 9 wherein the heat-stable DNA polymerase comprises a Taq DNA polymerase.

15. The material of claim 5 wherein the metal ions are selected from the group consisting of magnesium, manganese, calcium, cobalt, nickel, copper, zinc, iron, and combinations thereof.

16. The material of claim 5 wherein the metal ions comprise magnesium.

17. The material of claim 1 wherein the PCR reagent is encapsulated by the polylactone matrix.

18. The material of claim 1 wherein the PCR reagent is micro-encapsulated by the polylactone matrix.

19. A material for use in hot-start PCR comprising:

a polylactone matrix comprising a polylactone selected from the group consisting of poly(β

-propiolactone), poly(β

-butyrolactone), poly(γ

-butyrolactone), poly(α

-methyl-γ

-butyrolactone), poly(γ

-methyl-γ

-hexanolactone), poly(γ

-valerolactone), poly(γ

-caprolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof; and

a PCR reagent selected from the group consisting of oligonucleotides, deoxynucleoside triphosphates, polymerase enzymes, metal ions, and combinations thereof;

wherein the polylactone matrix substantially sequesters the PCR reagent from participation in a polymerase chain reaction at ambient temperature.

20. The material of claim 19 wherein:

the oligonucleotides are selected from the group consisting of antisense primers, sense primers, and a combination thereof;

the deoxynucleoside triphosphates are selected from the group consisting of dATP, dTTP, dCTP, dGTP, dUTP, dITP, and combinations thereof;

the polymerase enzymes comprise a heat-stable DNA polymerase; and

the metal ions are selected from the group consisting of magnesium, manganese, calcium, cobalt, nickel, copper, zinc, iron, and combinations thereof.

21. The material of claim 20 wherein the PCR reagent is encapsulated by the polylactone matrix.

22. The material of claim 20 wherein the PCR reagent is micro-encapsulated by the polylactone matrix.

23. A method of amplifying nucleic acid in a polymerase chain reaction comprising:

(a) providing a target nucleic acid sequence;

(b) combining the target nucleic acid sequence with a plurality of PCR reagents, wherein at least one of the plurality of PCR reagents is introduced in a polylactone matrix and is not available to participate in the polymerase chain reaction at ambient temperature; and

(c) heating the polylactone matrix to release the at least one of the plurality of PCR reagents therein.

24. The method of claim 23 wherein the polylactone matrix comprises a polylactone selected from the group consisting of poly(β

-propiolactone), poly(β

-butyrolactone), poly(γ

-butyrolactone), poly(α

-methyl-γ

-butyrolactone), poly(γ

-methyl-γ

-hexanolactone), poly(γ

-valerolactone), poly(γ

-caprolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof.

25. The method of claim 23 wherein the polylactone matrix comprises a polylactone selected from the group consisting of poly(β

-propiolactone), poly(γ

-butyrolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof.

26. The method of claim 23 wherein the polylactone matrix comprises poly(ε

-caprolactone).

27. The method of claim 23 wherein the PCR reagents are selected from the group consisting of oligonucleotides, deoxynucleoside triphosphates, polymerase enzymes, metal ions, and combinations thereof.

28. The method of claim 27 wherein the oligonucleotides are selected from the group consisting of antisense primers, sense primers, and a combination thereof.

29. The method of claim 27 the deoxynucleoside triphosphates are selected from the group consisting of dATP, dTTP, dCTP, dGTP, dUTP, dITP, and combinations thereof.

30. The method of claim 27 wherein the polymerase enzymes comprise a heat-stable DNA polymerase.

31. The method of claim 30 wherein the heat-stable DNA polymerase comprises an enzyme from a species selected from the group consisting of Thermus, Bacillus, Thermococcus, Thermotoga, Pyrococcus, and combinations thereof.

32. The method of claim 31 wherein the Thermus is selected from the group consisting of Thermus aquaticus, Thermus thermophilus, and a combination thereof.

33. The method of claim 31 wherein the Bacillus comprises Bacillus stearothermophilus.

34. The method of claim 31 wherein the Thermococcus comprises Thermococcus litoralis.

35. The method of claim 31 wherein the Thermotoga comprises Thermotoga maritama.

36. The method of claim 31 wherein the heat-stable DNA polymerase comprises a Taq DNA polymerase.

37. The method of claim 27 wherein the metal ions are selected from the group consisting of magnesium, manganese, calcium, cobalt, nickel, copper, zinc, iron, and combinations thereof.

38. The method of claim 27 wherein the metal ions comprise magnesium.

39. The method of claim 23 wherein the PCR reagent is encapsulated by the polylactone matrix.

40. The method of claim 23 wherein the PCR reagent is micro-encapsulated by the polylactone matrix.

41. A kit for hot-start PCR comprising:

a material comprising a polylactone matrix and a first PCR reagent;

wherein the polylactone matrix substantially sequesters the first PCR reagent from participation in a polymerase chain reaction at ambient temperature.

42. The kit of claim 41 wherein the polylactone matrix comprises a polylactone selected from the group consisting of poly(β

-propiolactone), poly(β

-butyrolactone), poly(γ

-butyrolactone), poly(α

-methyl-γ

-butyrolactone), poly(γ

-methyl-γ

-hexanolactone), poly(γ

-valerolactone), poly(γ

-caprolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof.

43. The kit of claim 41 wherein the polylactone matrix comprises a polylactone selected from the group consisting of poly(β

-propiolactone), poly(γ

-butyrolactone), poly(δ

-valerolactone), poly(ε

-caprolactone), poly(η

-heptanolactone), and combinations thereof.

44. The kit of claim 41 wherein the polylactone matrix comprises poly(ε

-caprolactone).

45. The kit of claim 41 wherein the first PCR reagent is selected from the group consisting of oligonucleotides, deoxynucleoside triphosphates, polymerase enzymes, metal ions, and combinations thereof.

46. The kit of claim 41 wherein the first PCR reagent comprises an oligonucleotide.

47. The kit of claim 46 wherein the oligonucleotide is selected from the group consisting of sense primers, antisense primers, and a combination thereof.

48. The kit of claim 46 further comprising a second PCR reagent selected from the group consisting of deoxynucleoside triphosphates, polymerase enzymes, metal ions, and combinations thereof.

49. The kit of claim 41 wherein the first PCR reagent comprises a polymerase enzyme.

50. The kit of claim 49 wherein the polymerase enzyme comprises a heat-stable DNA polymerase.

51. The kit of claim 50 wherein the heat-stable DNA polymerase comprises a Taq DNA polymerase.

52. The kit of claim 49 further comprising a second PCR reagent selected from the group consisting of oligonucleotides, deoxynucleoside triphosphates, metal ions, and combinations thereof.

53. The kit of claim 41 wherein the first PCR reagent comprises a metal ion.

54. The kit of claim 53 wherein the metal ion comprises magnesium.

55. The kit of claim 53 further comprising a second PCR reagent selected from the group consisting of oligonucleotides, deoxynucleoside triphosphates, polymerase enzymes, and combinations thereof.