Late-PCR

• US 20080280292A1
• Filed: 02/02/2007
• Published: 11/13/2008
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Rice, John, Wangh, Lawrence J., Hartshorn, Cristina, Sanchez, J. Aquiles, Pierce, Kenneth
• Granted Patent: US 8,367,325 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

A non-symmetric polymerise chain reaction (PCR) amplification method employing a limiting primer in low concentration whose concentration-adjusted melting point at least equals, and preferably exceeds, that of the excess primer, the latter in turn not being more than 25° C. below the melting temperature of the amplicon. Assays employing such amplification and labeled hybridization probes, including assays that include a detection step following primer extension or a low-temperature probe, or both. Kits for performing such assays and primer or primer-and-probe sets for performing the foregoing amplifications and assays.

Claims

1. An oligonucleotide set comprising a pair of primers for amplifying a selected DNA sequence by a non-symmetric polymerase chain reaction (PCR) comprising a first amount of a limiting primer used at a concentration of not more than 200 mM and a second amount, at least five times greater than the first amount, of an excess primer used at a concentration at least five times greater than the limiting primer concentration, wherein the concentration-adjusted melting temperature of the limiting primer is equal to or greater than the concentration-adjusted melting point of the excess primer when the concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, and wherein the melting temperature of an amplicon produced by extension of the excess primer does not exceed the concentration-adjusted melting temperature of the excess primer by more than 18°

C.

2. The oligonucleotide set according to claim 1, wherein the concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the concentration-adjusted melting temperature of the excess primer.

3. The oligonucleotide set according to claim 1, wherein the second amount is at least ten times greater than the first amount and wherein the excess primer is used at a concentration at least ten times greater than the limiting primer concentration.

4. The oligonucleotide set according to claim 1, wherein the melting point of the amplicon exceeds the concentration-adjusted melting temperature of the excess primer by 7-15°

C.

5. The oligonucleotide set according to claim 2, wherein the melting temperature of the amplicon exceeds the concentration-adjusted melting temperature of the excess primer by 7-15°

C.

6. The oligonucleotide set according to claim 3, wherein the melting temperature of the amplicon exceeds the concentration-adjusted melting temperature of the excess primer by 7-15°

C.

45. The oligonucleotide set according to claim 1, wherein the formula is:

T_m=Δ

H/(Δ

S+R ln(C/2))−

273.15+12 log [M], wherein

Δ

H is enthalpy,

Δ

S is entropy,

C is concentration of the primer,

R is universal gas constant, and

[M] is molar concentration of monovalent cations.

7. An oligonucleotide set for amplifying a selected DNA sequence by a polymerase chain reaction (PCR) comprising a pair of matched PCR limiting primers for producing a first amplicon and an excess primer for producing a second, single-stranded amplicon utilizing the first amplicon as a template, wherein (i) each of the limiting primers is present in a first amount and is used at a concentration not exceeding 200 nM, (i) the excess primer is present in a second amount, at least five times greater than the first amount, and is used at a concentration at least five times higher than the concentration of each limiting primer, and (iii) wherein a concentration-adjusted melting temperature of the excess primer is at least 5°

C. lower than a concentration-adjusted melting temperature of both limiting primers when the concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition.

8. The oligonucleotide set according to claim 7, wherein the melting temperature of the second amplicon exceeds the concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

9. The oligonucleotide set according to claim 7, wherein the second amount is at least ten times greater than the first amount and wherein the excess primer is used at a concentration at least ten times greater than the limiting primer concentration.

46. The oligonucleotide set according to claim 7, wherein the formula is:

T_m=Δ

H/(Δ

S+R ln(C/2))−

273.15+12 log [M], wherein

Δ

H is enthalpy,

Δ

S is entropy,

C is concentration of the primer,

R is universal gas constant, and

[M] is molar concentration of monovalent cations.

10. An oligonucleotide set for a homogenous, non-symmetric polymerase chain reaction (PCR) amplification assay for a selected DNA sequence comprising (i) a first amount of a limiting primer used at a concentration not exceeding 200 nM, (ii) a second amount, at least five times greater than the first amount, of an excess primer used at a concentration at least five times greater than the limiting primer concentration, and (iii) a labeled hybridization probe that emits a signal indicative of the presence of a product produced by extension of the excess primer, wherein the concentration-adjusted melting temperature of the limiting primer is at least equal to the concentration-adjusted melting temperature of the excess primer when the concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, and wherein the melting point of an amplicon produced by the primers exceeds the concentration-adjusted melting point of the excess primer by not more than 25°

C.

11. The oligonucleotide set according to claim 10, wherein the melting temperature of the amplicon exceeds the concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

12. The oligonucleotide set according to claim 10, wherein the concentration-adjusted melting temperature of the limiting primer exceeds the concentration-adjusted melting temperature of the excess primer by at least 3°

C.

13. The oligonucleotide set according to claim 10, wherein the second amount is at least ten times greater than the first amount and wherein the excess primer is used at a concentration at least ten times greater than the limiting primer concentration.

14. The oligonucleotide set according to claim 10, wherein the probe hybridizes to an extension product of the excess primer and signals upon hybridization.

15. The oligonucleotide set according to claim 14, wherein the probe comprises a first hybridization probe specific for a first allele and a second hybridization probe specific for a second allele.

16. The oligonucleotide set according to claim 14, wherein the hybridization probe is a molecular beacon probe.

17. The oligonucleotide set according to claim 14, wherein the probe is used at a probe concentration and wherein a probe concentration-adjusted melting temperature of the probe is at least 5°

C. below the concentration-adjusted melting point of the limiting primer when the concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition.

18. The oligonucleotide set according to claim 17, wherein the probe concentration-adjusted melting point of the probe is at least 10°

C. below the concentration-adjusted melting point of the limiting primer when the concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition.

19. The oligonucleotide set according to claim 18, wherein the probe is a molecular beacon probe.

47. The oligonucleotide set according to claim 10, wherein the formula is:

T_m=Δ

H/(Δ

S+R ln(C/2))−

273.15+12 log [M], wherein

Δ

H is enthalpy,

Δ

S is entropy,

C is concentration of the primer,

R is universal gas constant, and

[M] is molar concentration of monovalent cations.

20. A kit of reagents for performing a homogeneous polymerase chain reaction assay for at least one pre-selected DNA target sequence, comprising at least one pair of polymerase chain reaction primers including a first primer and a second primer, four deoxyribonucleotide triphosphates, a thermostable DNA polymerase, and a labeled hybridization probe that emits a detectable signal upon hybridization, wherein

a) the T_m of the first primer is at least 5°

C. greater than the T_m of the second primer and the concentration of the second primer is at least ten times greater than the concentration of the first primer, and

b) the labeled hybridization probe binds to an extension product of the second primer.

21. The kit according to claim 20, wherein the T_m of the first primer is 10°

C.-20°

C. greater than the T_m of the second primer.

22. The kit according to claim 21, wherein the concentration of the second primer is 20-100 times greater than the concentration of the first primer.

23. The kit according to claim 20, wherein the concentration of the second primer is 20-100 times greater than the concentration of the first primer.

24. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each target sequence, a first amount of a limiting primer to be used at a concentration of up to 200 nM, a second amount, at least five times greater than the first amount, of an excess primer to be used at a concentration at least give times greater than the limiting primer concentration, and a labeled hybridization probe that emits a signal indicative of the presence of a product produced by extension of the excess primer, wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least equal to the initial concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, and wherein the melting temperature of an amplicon produced by the primers exceeds the initial concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

25. The kit according to claim 24 for performing a multiplex assay for at least two target sequences, wherein the initial, concentration-adjusted melting temperatures of all limiting primers are at least equal to the initial concentration-adjusted melting temperatures of all excess primers.

26. The kit according to claim 24, wherein the initial concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

27. The kit according to claim 24, wherein the probe hybridizes to an extension product of the excess primer and signals upon hybridization.

28. The kit according to claim 27, wherein the probe comprises a first probe for one allelic variant and a second probe for a second allelic variant.

29. The kit according to claim 27 for performing a multiplex assay for at least two target sequences, wherein the initial, concentration-adjusted melting temperatures of all limiting primers are at least equal to the initial concentration-adjusted melting temperatures of all excess primers.

30. The kit according of claim 27, wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least 3°

C. higher than the initial, concentration-adjusted melting temperature of the excess primer.

31. The kit according to claim 27 wherein the initial, concentration-adjusted melting temperature of the probe is at least 5°

C. lower than the initial, concentration-adjusted melting temperature of the limiting primer.

32. The kit according to claim 31 wherein the initial, concentration-adjusted melting temperature of the probe is at least 110°

C. lower than the initial, concentration-adjusted melting temperature of the limiting primer.

33. The kit according to claim 27, further comprising directions specifying a detection step following primer annealing for at least some PCR cycles and specifying an annealing temperature or temperatures to be used for annealing the limiting primer, wherein the initial, concentration-adjusted melting temperature of the probe is at least 5°

C. below the mean of the annealing temperature or temperatures when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial probe concentration and composition.

34. The kit according to claim 33, wherein the initial, concentration-adjusted melting temperature of the probe is at least 10°

C. below the mean of the annealing temperature or temperatures.

35. The kit according to claim 33, wherein the melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°

C.

36. The kit according to claim 35, wherein the concentration-adjusted melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by 7-15°

C.

48. The kit according to claim 24, wherein the formula is:

T_m=Δ

H/(Δ

S+R ln(C/2))−

273.15+12 log [M], wherein

Δ

H is enthalpy,

Δ

S is entropy,

C is concentration of the primer,

R is universal gas constant, and

[M] is molar concentration of monovalent cations.

37. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each target sequence, a pair of matched PCR limiting primers for producing a first amplicon, an excess primer for producing a second, single-stranded amplicon utilizing the first amplicon as a template, and a labeled hybridization probe that emits a signal indicative of the presence of the second amplicon, wherein the limiting primers are to be used in a first, equimolar, amount at a concentration of up to 200 nM, the excess primer is to be used in a second amount, at least five times greater than the first amount, and at a concentration at least five times greater than the concentration of the limiting primers, and wherein an initial, concentration-adjusted melting temperature of the excess primer is at least 5°

C. lower than an initial, concentration-adjusted melting temperatures of the limiting primers when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial probe concentration and composition.

38. The kit according to claim 37, wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

39. The kit according to claim 37, wherein the second amount is at least ten times greater than the first amount and wherein the excess primer is to be used at a concentration at least ten times greater than the limiting primer'"'"'s concentration.

49. The oligonucleotide set according to claim 37, wherein the formula is:

T_m=Δ

H/(Δ

S+R ln(C/2))−

273.15+12 log [M], wherein

Δ

H is enthalpy,

Δ

S is entropy,

C is concentration of the primer,

R is universal gas constant, and

[M] is molar concentration of monovalent cations.

40. An oligonucleotide set comprising a pair of primers for amplifying a selected DNA sequence by a non-symmetric polymerase chain reaction (PCR) comprising (a) a first amount of a limiting primer used at a concentration of not more than 200 nM and (b) a second amount, at least five times greater than the first amount, of an excess primer used at a concentration at least five times greater than the limiting primer concentration, wherein the concentration-adjusted melting temperature of the limiting primer is equal to or greater than the concentration-adjusted melting point of the excess primer when the concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, and wherein the melting temperature of an amplicon produced by extension of the excess primer does not exceed the concentration-adjusted melting temperature of the excess primer by more than 18°

C.

41. An oligonucleotide set for amplifying a selected DNA sequence by a polymerase chain reaction (PCR) comprising a pair of matched PCR limiting primers for producing a first amplicon and an excess primer for producing a second, single-stranded amplicon utilizing the first amplicon as a template, wherein (i) each of the limiting primers is present in a first amount and is used at a concentration not exceeding 200 nM, (i) the excess primer is present in a second amount, at least five times greater than the first amount, and is used at a concentration at least five times higher than the concentration of each limiting primer, and (iii) wherein a concentration-adjusted melting temperature of the excess primer is at least 5°

C. lower than a concentration-adjusted melting temperature of both limiting primers when the concentration-adjusted melting temperature is determined using a Nearest Neighbor formula.

42. An oligonucleotide set for a homogenous, non-symmetric polymerase chain reaction (PCR) amplification assay for a selected DNA sequence comprising (i) a first amount of a limiting primer used at a concentration not exceeding 200 μ

M, (ii) a second amount, at least five times greater than the first amount, of an excess primer used at a concentration at least five times greater than the limiting primer concentration, and (iii) a labeled hybridization probe that emits a signal indicative of the presence of a product produced by extension of the excess primer, wherein the concentration-adjusted melting temperature of the limiting primer is at least equal to the concentration-adjusted melting temperature of the excess primer when the concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, and wherein the melting point of an amplicon produced by the primers exceeds the concentration-adjusted melting point of the excess primer by not more than 25°

C.

43. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each target sequence, a first amount of a limiting primer to be used at a concentration of up to 200 nM, a second amount, at least five times greater than the first amount, of an excess primer to be used at a concentration at least give times greater than the limiting primer concentration, and a labeled hybridization probe that emits a signal indicative of the presence of a product produced by extension of the excess primer, wherein the initial, concentration-adjusted melting temperature of the limiting primer is at least equal to the initial concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, and wherein the melting temperature of an amplicon produced by the primers exceeds the initial concentration-adjusted melting temperature of the excess primer by not more than 25°

C.

44. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each target sequence, a pair of matched PCR limiting primers for producing a first amplicon, an excess primer for producing a second, single-stranded amplicon utilizing the first amplicon as a template, and a labeled hybridization probe that emits a signal indicative of the presence of the second amplicon, wherein the limiting primers are to be used in a first, equimolar, amount at a concentration of up to 200 nM, the excess primer is to be used in a second amount, at least five times greater than the first amount, and at a concentration at least five times greater than the concentration of the limiting primers, and wherein an initial, concentration-adjusted melting temperature of the excess primer is at least 5°

C. lower than an initial, concentration-adjusted melting temperatures of the limiting primers when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula.