Late-PCR

• US 8,367,325 B2
• Filed: 02/02/2007
• Issued: 02/05/2013
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Wangh, Lawrence J., Pierce, Kenneth, Hartshorn, Cristina, Rice, John, Sanchez, J. Aquiles
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US11/701,428
• Publication Number:
  US 20080280292A1
• Time in Patent Office: 2,195 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 polymerase chain reaction (PCR) primers in a single buffer for amplifying a selected DNA amplification target sequence, said target sequence and primers defining a double-stranded amplification sequence bracketed by the primer pair, said amplification sequence having a melting temperature (T_m^A), wherein T_m^A=81.5+0.41(% G+% C)−

500/L+16.6 log 0.07/(1+0.7(0.07)), where L is the length in nucleotides, comprising a limiting primer and an excess primer,

wherein the ratio (X) of limiting primer to excess primer is not more than 0.05, wherein X=(concentration of limiting primer)/(concentration of excess primer);

wherein the limiting primer has a melting temperature (T_m^L), wherein T_m^L=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, and the excess primer has a melting temperature (T_m^X), wherein T_m^X=, Δ

H/(Δ

S+R ln(10<sup>−

6</sup>/2))−

273.15+12 log 0.07, wherein the difference between T_m^L and T_m^X is equal to or higher than 0°

C. and, if the limiting primer is not fully complementary to the amplification target sequence, the melting temperature of the imperfect hybrid between the limiting primer and the amplification target sequence (T_m^LA), wherein T_m^LA=Δ

H/(Δ

S+R ln (X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, is not more than 5°

C. below the melting temperature of the excess primer; and

wherein the melting temperature of the amplification sequence does not exceed the melting temperature of the excess primer by more than 18°

C., where R is the universal gas constant, and where Δ

H is enthalpy and Δ

S is entropy.

2. The oligonucleotide set according to claim 1 wherein the melting temperature of the limiting primer exceeds the melting temperature of the excess primer by at least 3°

C.

3. The oligonucleotide set according to claim 2 wherein the melting temperature of the amplification sequence exceeds the melting temperature of the excess primer by 7-15°

C.

4. The oligonucleotide set according to claim 1 wherein the melting temperature of the amplification sequence exceeds the melting temperature of the excess primer by 7-15°

C.

5. An oligonucleotide set comprising a pair of polymerase chain reaction (PCR) primers in a single buffer for amplifying a selected DNA amplification target sequence, said target sequence and primers defining a double-stranded amplification sequence bracketed by the primer pair, said amplification sequence having a melting temperature (T_m^A), wherein T_m^A=81.5+0.41(% G+% C)500/L+16.6 log 0.07/(1+0.7(0.07)), where L is the length in nucleotides, comprising a limiting primer and an excess primer,

wherein the ratio (X) of limiting primer to excess primer is not more than 0.2, wherein X=(concentration of limiting primer)/(concentration of excess primer);

wherein the limiting primer has a melting temperature (T_m^L), wherein T_m^L=Δ

H/(Δ

S+R ln (X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, and the excess primer has a melting temperature (T_m^X), wherein T_m^X=Δ

H/(Δ

S+R ln(10<sup>−

6</sup>/2))−

273.15+12 log 0.07, wherein T_m^L exceeds T_m^X by at least 3°

C. and, if the limiting primer is not fully complementary to the amplification target sequence, the melting temperature of the imperfect hybrid between the limiting primer and the amplification target sequence (T_m^LA), wherein T_m^LA=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, is not more than 5°

C. below the melting temperature of the excess primer; and

wherein the melting temperature of the amplification sequence does not exceed the melting temperature of the excess primer by more than 18°

C., where R is the universal gas constant, and where Δ

H is enthalpy and Δ

S is entropy.

6. The oligonucleotide set according to claim 5, wherein X is not more than 0.1.

7. An oligonucleotide set comprising, in a single buffer,

a) a pair of polymerase chain reaction (PCR) primers in a single buffer for amplifying a selected DNA amplification target sequence, said amplification target sequence and said primers defining a double-stranded amplification sequence bracketed by the primer pair, said amplification sequence having a melting temperature (T_m^A), wherein T_m^A=81.5+0.41(% G+% C)−

500/L+16.6 log 0.07/(1+0.7(0.07)), where L is the length in nucleotides, comprising a limiting primer and an excess primer, wherein the concentration ratio (X) of limiting primer to excess primer is not more than 0.2, wherein X=(concentration of limiting primer)/(concentration of excess primer); and

b) at least one labeled hybridization probe consisting essentially of conventional nucleotides that hybridizes to the same strand of the amplification sequence as does the limiting primer and that emits a detectable signal upon hybridization, wherein Y is the concentration ratio of the probe to the excess primer, wherein Y=(concentration of probe)/(concentration of excess primer) and wherein the melting temperature of the at least one probe-amplification sequence hybrid (T_m^PA) measured at a concentration of Y μ

M is at least 5°

C. below the melting temperature of the limiting primer (T_m^L), wherein T_m^L=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, where R is the universal gas constant, and where Δ

H is enthalpy and Δ

S is entropy.

8. The oligonucleotide set according to claim 7 wherein the difference between T_m^L and the melting temperature of the excess primer (T_m^X), wherein T_m^X=Δ

H/(Δ

S+R ln(10<sup>−

6</sup>/2))−

273.15+12 log 0.07, is equal to or higher than 0°

C. and, if the limiting primer is not fully complementary to the amplification target sequence, the melting temperature of the imperfect hybrid between the limiting primer and the amplification target sequence (T_m^LA), wherein T_m^LA=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, is not more than 5°

C. below the melting temperature of the excess primer.

9. The oligonucleotide set according to claim 8, wherein the melting temperature of the limiting primer exceeds the melting temperature of the excess primer by at least 3°

C.

10. The oligonucleotide set according to claim 8, wherein X is not more than 0.1.

11. The oligonucleotide set according to claim 8, wherein the melting temperature of the amplification sequence exceeds the melting temperature of the excess primer by not more than 18°

C.

12. The oligonucleotide set according to claim 11, wherein the melting temperature of the at least one probe-amplification sequence hybrid is at least 10°

C. below the melting temperature of the limiting primer.

13. The oligonucleotide set according to claim 8, wherein said at least one probe comprises a first hybridization probe specific for a first allele and a second hybridization probe specific for a second allele.

14. The oligonucleotide set according to claim 8, wherein the melting temperature of the at least one probe-amplification sequence hybrid is at least 10°

C. below the melting temperature of the limiting primer.

15. The oligonucleotide set according to claim 7, wherein said at least one 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 7, wherein said at least one hybridization probe is a DNA probe.

17. The oligonucleotide set according to claim 7, wherein the melting temperature of the at least one probe-amplification sequence hybrid is at least 10°

C. below the melting temperature of the limiting primer.

18. An oligonucleotide set comprising, in a single buffer,

a) a pair of polymerase chain reaction (PCR) primers in a single buffer for amplifying a selected DNA amplification target sequence, said amplification target sequence and said primers defining a double-stranded amplification sequence bracketed by the primer pair, said amplification sequence having a melting temperature (T_m^A), wherein T_mA=81.5+0.41(% G+% C)−

500/L+16.6 log 0.07/(1+0.7(0.07)), where L is the length in nucleotides, comprising a limiting primer and an excess primer, wherein the concentration ratio (X) of limiting primer to excess primer is not more than 0.2, wherein X=(concentration of limiting primer)/(concentration of excess primer); and

b) at least one labeled hybridization probe that hybridizes to the same strand of the amplification sequence as does the limiting primer and that emits a detectable signal upon hybridization, wherein Y is the concentration ratio of the probe to the excess primer, wherein Y=(concentration of probe)/(concentration of excess primer), and wherein the melting temperature of the at least one probe-amplification sequence hybrid measured at a concentration of Y μ

M is at least 10°

C. below the melting temperature of the limiting primer (T_m^A), wherein T_m^A=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, where R is the universal gas constant, and where Δ

H is enthalpy and Δ

S is entropy.

30. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA amplification target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each amplification target sequence, an oligonucleotide set according to claim 18.

19. A kit of reagents for performing a homogenous polymerase chain reaction (PCR) assay for at least one pre-selected DNA amplification target sequence comprising a thermostable DNA polymerase, dNTP'"'"'s and, for each amplification target sequence, an oligonucleotide set comprising, in a single buffer,

a) a pair of polymerase chain reaction (PCR) primers in a single buffer for amplifying a selected DNA amplification target sequence, said amplification target sequence and said primers defining a double-stranded amplification sequence bracketed by the primer pair, said amplification sequence having a melting temperature (T_m^A), wherein T_m^A=81.5+0.41(% G+% C)−

500/L+16.6 log 0.071(1+0.7(0.07)), where L is the length in nucleotides, comprising a limiting primer and an excess primer, wherein the concentration ratio (X) of limiting primer to excess primer is not more than 0.2, wherein X=(concentration of limiting primer)/(concentration of excess primer); and

b) at least one labeled hybridization probe consisting essentially of conventional nucleotides that hybridizes to the same strand of the amplification sequence as does the limiting primer and that emits a detectable signal upon hybridization, wherein Y is the concentration ratio of the probe to the excess primer, wherein Y=(concentration of probe)/(concentration of excess primer), and wherein the melting temperature of the at least one probe-amplification sequence hybrid measured at a concentration of Y μ

M is at least 5°

C. below the melting temperature of the limiting primer (T_m^L), wherein T_m^L=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, where R is the universal gas constant, and where Δ

H is enthalpy and Δ

S is entropy.

20. The kit according to claim 19 wherein the difference between T_m^L and the melting temperature of the excess primer (T_m^X), wherein T_m^X=Δ

H/(Δ

S+R ln(10<sup>−

6</sup>/2))−

273.15+12 log 0.07, is equal to or higher than 0°

C. and, if the limiting primer is not fully complementary to the amplification target sequence, the melting temperature of the imperfect hybrid between the limiting primer and the amplification target sequence (T_m^LA), wherein T_m^LA=Δ

H/(Δ

S+R ln(X*10<sup>−

6</sup>/2))−

273.15+12 log 0.07, is not more than 5°

C. below the melting temperature of the excess primer.

21. The kit according to claim 20, wherein the melting temperature of the limiting primer exceeds the melting temperature of the excess primer is at least 3°

C.

22. The kit according to claim 21, wherein:

X is not more than 0.05.

23. The kit according to claim 20, wherein the melting temperature of the amplification sequence exceeds the melting temperature of the excess primer by not more than 18°

C.

24. The kit according to claim 23, wherein the melting temperature of the at least one probe-amplification sequence hybrid is at least 10°

C. below the melting temperature of the limiting primer.

25. The kit according to claim 20, wherein said at least one probe comprises a first hybridization probe specific for a first allele and a second hybridization probe specific for a second allele.

26. The kit according to claim 20, wherein the melting temperature of the at least one probe-amplification sequence hybrid is at least 10°

C. below the melting temperature of the limiting primer.

27. The kit according to claim 19, wherein said at least one probe comprises a first hybridization probe specific for a first allele and a second hybridization probe specific for a second allele.

28. The kit according to claim 19, wherein said at least one hybridization probe is a DNA probe.

29. The kit according to claim 28, wherein the melting temperature of the at least one probe-amplification sequence hybrid is at least 10°

C. below the melting temperature of the limiting primer.