Patent ID: 8367325

Claim:
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.