Reagents and methods for improving reproducibility and reducing mispriming in PCR amplification

• US 20090226973A1
• Filed: 03/20/2009
• Published: 09/10/2009
• Est. Priority Date: 10/18/2004
• Status: Abandoned Application

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Reis, Arthur, Rice, John, Wangh, Lawrence J., Salk, Jesse, Hartshorn, Cristina, Sanchez, J. Aquiles, Pierce, Kenneth
• Application Number:
  US12/382,670
• Publication Number:
  US 20090226973A1
• Time in Patent Office: Days
• US Class Current: 435/91.200
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation

Abstract

An additive for preventing mispriming in polymerase chain reaction (PCR) amplifications and assays comprising a hairpin oligonucleotide having a stem duplex greater than six nucleotides in length and a stabilized stem terminus. The additive improves PCR amplifications, including LATE-PCR amplifications when added to initial amplification reaction mixtures. It can be included in oligonucleotide sets and in kits for PCR amplification and assays.

Claims

1. A reagent capable of preventing at least one manifestation of mispriming in a polymerase chain reaction (PCR) amplification to produce at least one amplified DNA product when added at a concentration of not more than 650 nM to a PCR amplification mixture that includes 1.25 units of a thermostable DNA polymerase per 25 μ

l of reaction mixture, said reagent being a non-extendable oligonucleotide that has a stem-loop structure, that is not a hybridization probe for said at least one amplified DNA product, that has a stem that is greater than six nucleotides in length, that is stabilized at its terminus away from the loop, and that has a calculated stem melting temperature (Tm) below 94°

C.

2. The reagent according to claim 1 wherein the stem is terminally stabilized by interacting chemical moieties covalently attached to the 3′

and 5′

nucleotides of the stem.

3. The reagent according to claim 2 wherein the interacting chemical moieties are non-fluorescent quencher moieties.

4. The reagent according to claim 3 wherein the loop is an oligonucleotide comprising at least three nucleotides.

5. The reagent according to claim 4 wherein the stem comprises a double-stranded sequence of 9-12 base pairs.

6. The reagent according claim 3 wherein the stem comprises a double-stranded region of 9-12 base pairs.

7. The reagent according to claim 2 wherein the stem has a calculated melting temperature (Tm) is in the range of 72-85°

C.

8. The reagent according to claim 2 wherein the stem has a calculated melting temperature (Tm) in the range of 50-71°

C.

9. The reagent according to claim 2 wherein the stem comprises a double-stranded region of 9-12 base pairs.

10. The reagent according to claim 1 wherein the loop comprises an oligonucleotide comprising at least three nucleotides.

11. The reagent according to claim 10 wherein the stem comprises a double-stranded region of 9-12 base pairs.

12. The reagent according to claim 1 wherein the loop is a non-nucleotide chemical linker.

13. The reagent according to claim 12 wherein the chemical linker is a hydrocarbon chain.

14. The reagent according to claim 13 wherein the hydrocarbon chain is a methylene chain of 3-6 carbon atoms.

15. The reagent according to claim 13 wherein the stem is terminally stabilized by interacting chemical moieties covalently attached to the 3′

and 5′

nucleotides of the stem.

16. The reagent according to claim 15 wherein the interacting chemical moieties are non-fluorescent quencher moieties.

17. The reagent according to claim 15 wherein the calculated melting temperature of the stem is at least 50°

C.

18. The reagent according to claim 1 wherein the stem is stabilized by inclusion therein of non-natural terminal nucleotides.

19. The reagent according to claim 18 wherein the loop is an oligonucleotide comprising at least three nucleotides.

20. The reagent according to claim 18 wherein the calculated melting temperature of the stem is at least 50°

C.

21. The reagent according to claim 20 wherein the stem comprises a double-stranded region of 9-12 base pairs.

22. The reagent according to claim 18 wherein the loop is a non-nucleotide chemical linker.

23. The reagent according to claim 1 wherein the calculated melting temperature of the stem is at least 50°

C.

24. The reagent according to claim 23 wherein the calculated melting temperature of the stem is in the range of 72-85°

C.

25. A method for preventing at least one manifestation of mispriming in a polymerase chain reaction (PCR) amplification of a reaction mixture that includes a thermostable DNA polymerase, comprising adding to said reaction mixture at least one reagent according to claim 1.

26. The method of claim 25 wherein said reagent has a stem that is terminally stabilized by interacting chemical moieties covalently attached to the 3′

and 5′

nucleotides of the stem.

27. The method of claim 26 wherein said reagent has an oligonucleotide loop comprising at least three nucleotides and said reagent is added to the reaction mixture at a concentration of not more than 300 nM.

28. The method of claim 26 wherein said reagent has a stem of 9-12 base pairs in length.

29. The method of claim 36 wherein said reagent has a loop that is a non-nucleotide chemical linker.

30. The method of claim 29 wherein said reagent is added to the reaction mixture at a concentration of not more than 1000 nM.

31. The method of claim 29 wherein said linker is an alkylene chain.

32. The method of claim 29 wherein said linker comprises a chain of 3-6 carbon atoms.

33. The method of claim 25 wherein said reagent has an oligonucleotide loop comprising at least three nucleotides.

34. The method of claim 25 wherein said reagent has a loop that is a non-nucleotide chemical linker.

35. The method of claim 25 wherein the PCR amplification comprises a liner-after-the exponential PCR (LATE-PCR) assay including a low-temperature detection step.

36. The method of claim 25 wherein the PCR amplification is followed by end-point detection.

37. A kit of reagents for performing a polymerase chain reaction (PCR) amplification comprising a thermostable DNA polymerase, at least one pair of PCR primers, dNTP'"'"'s and at least one reagent according to claim 1.

38. The kit according to claim 37 further comprising reagents for nucleic acid isolation.

39. The kit according to claim 37 wherein the reagent has an oligonucleotide loop of at least three nucleotides.

40. The kit according to claim 37 wherein the reagent has a loop that is a non-nucleotide chemical linker.

41. The kit according to claim 37 wherein the reagent has a stem that is terminally stabilized by interacting chemical moieties covalently attached to the 3′

and 5′

nucleotides of the stem.

42. A set of oligonucleotides for performing a polymerase chain reaction (PCR) amplification comprising at least one pair of PCR primers and at least one reagent according to claim 1.

43. The set according to claim 42 further including at least one fluorescently labeled probe that hybridizes to the amplified product defined by said at least one pair of PCR primers.