A method of amplifying target polynucleotides, the method comprising the steps of:
- providing a first target polynucleotide, a second target polynucleotide, and a third target polynucleotide, such that the first target polynucleotide and only the first target polynucleotide has an amplicon melting temperature above a predetermined amplicon melting temperature, the second target polynucleotide and only the second target polynucleotide has a primer annealing temperature below a predetermined primer annealing temperature, and the third target polynucleotide and only the third target polynucleotide has a replication time above a predetermined polymerase extension time;
- amplifying in a first stage reaction using a nucleic acid polymerase the first, second, and third target polynucleotides in the presence of three distinct fluorescent indicators, each fluorescent indicator generating a distinct optical signal indicating quantity of amplification products of the first, second, and third target polynucleotides, respectively, and the first-stage reaction having an amplicon denaturation temperature above the predetermined amplicon melting temperature, a primer annealing temperature below the predetermined primer annealing temperature, and a polymerase extension time above the predetermined polymerase extension time, such that each of the first, second, and third target polynucleotides is amplified in the first stage reaction;
- monitoring the optical signals of the fluorescent indicators;
- and
- initiating a second stage reaction by changing the reaction condition of the first stage reaction as follows and in any order:
- (1) whenever the optical signal of the fluorescent indicator indicating the quantity of the amplification product of the first target polynucleotide reaches or exceeds a predetermined level, reducing the amplicon denaturation temperature of the reaction to below the predetermined amplicon melting temperature but above each of the amplicon melting temperatures of the second and third target polynucleotides, thereby terminating amplification of the first target polynucleotide,
- (2) whenever the optical signal of the fluorescent indicator indicating the quantity of the amplification product of the second target polynucleotide reaches or exceeds a predetermined level, increasing the primer annealing temperature of the reaction to above the predetermined primer annealing temperature but below each of the primer annealing temperatures of the first and third target polynucleotides, thereby terminating amplification of the second target polynucleotide, and
- (3) whenever the optical signal of the fluorescent indicator indicating the quantity of the amplification product of the third target polynucleotide reaches or exceeds a predetermined level, reducing the polymerase extension time of the reaction to below the predetermined polymerase extension time but above each of the replication times of the first and second target polynucleotides, thereby terminating amplification of the third target polynucleotide.