Multi-stage amplification reactions by control of sequence replication times

A method of successively amplifying target polynucleotides in a polymerase chain reaction, the method comprising the steps of:

• providing at least one first target polynucleotide, at least one second target polynucleotide, and at least one 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 melting temperature below a predetermined primer melting 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 the first, second, and third target polynucleotides in the presence of distinct fluorescent indicators for each thereof, each fluorescent indicator being capable of generating a distinct optical signal related to a quantity 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 melting temperature, and a polymerase extension time of a first value that permits the third target polynucleotide to be replicated;
• monitoring the optical signals of the fluorescent indicators;
• and
• successively changing (i) the amplicon melting temperature to a value below that of the predetermined amplicon denaturation temperature, (ii) the primer melting temperature to a value above that of the predetermined primer melting temperature, or (iii) the polymerase extension time to a second value less than the replication time of the third target polynucleotide to initiate a successive reaction stage, whenever the optical signal of a fluorescent indicator reaches or exceeds a predetermined level, so that first, second, and third target polynucleotides are successively amplified.

A kit for sequential multiplex amplification of at least two target polynucleotides in a temperature-cycling amplification reaction having a polymerase extension step, the kit comprising:

• a first primer pair specific for a first target polynucleotide, the first primer pair defining a first amplicon;
• a second primer pair specific for a second target polynucleotide, the second primer pair defining a second amplicon;
• and
• at least one blocking oligonucleotides specific for the first amplicon, the at least one blocking oligonucleotide having a Tm and the at least one blocking oligonucleotide being selected so that whenever the polymerase extension step is carried out at a first temperature greater than the Tm, both the first and second amplicons are produced, and whenever the polymerase extension step is carried out at a second temperature less than the Tm, substantially only the second amplicon is produced.