Late-PCR
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.
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 =ΔH/(ΔS+R ln(X*10 −6 /2))−273.15+12 log 0.07, and the excess primer has a melting temperature (T m X ), wherein T m X =, ΔH/(ΔS+R ln(10 −6 /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 =ΔH/(ΔS+R ln (X*10 −6 /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 ΔH is enthalpy and ΔS 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 =ΔH/(ΔS+R ln (X*10 −6 /2))−273.15+12 log 0.07, and the excess primer has a melting temperature (T m X ), wherein T m X =ΔH/(ΔS+R ln(10 −6 /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 =ΔH/(ΔS+R ln(X*10 −6 /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 ΔH is enthalpy and ΔS 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 μM is at least 5° C. below the melting temperature of the limiting primer (T m L ), wherein T m L =ΔH/(ΔS+R ln(X*10 −6 /2))−273.15+12 log 0.07, where R is the universal gas constant, and where ΔH is enthalpy and ΔS 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 =ΔH/(ΔS+R ln(10 −6 /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 =ΔH/(ΔS+R ln(X*10 −6 /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 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 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 μM is at least 10° C. below the melting temperature of the limiting primer (T m A ), wherein T m A =ΔH/(ΔS+R ln(X*10 −6 /2))−273.15+12 log 0.07, where R is the universal gas constant, and where ΔH is enthalpy and ΔS is entropy.
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 μM is at least 5° C. below the melting temperature of the limiting primer (T m L ), wherein T m L =ΔH/(ΔS+R ln(X*10 −6 /2))−273.15+12 log 0.07, where R is the universal gas constant, and where ΔH is enthalpy and ΔS 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 =ΔH/(ΔS+R ln(10 −6 /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 =ΔH/(ΔS+R ln(X*10 −6 /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.
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 .