Restriction mediated quantitative polymerase chain reactions

A method of performing Polymerase Chain Reaction (PCR) comprising amplifying a template comprising a target sequence with a PCR nucleic acid amplification mixture comprising:

• 1) a template comprising a target sequence;
• 2) a Polymerase Chain Reaction (PCR) primer comprising the following general structure:
• wherein:
• 5′ Acceptor:
• represents one member of a fluorescence resonance energy transfer (FRET) pair;
• A:
• represents a nucleic acid sequence motif of 10 to 30 bases;
• 3-C:
• represents a linker region comprised of at least three carbon atoms;
• B:
• represents a double stranded restriction enzyme recognition site;
• A′:
• represents a nucleic acid sequence motif of 10 to 30 bases being complementary to the nucleic acid sequence motif of A;
• and
• R-prim:
• represents a nucleic acid sequence complementary to a target sequence in a nucleic acid sequence to be amplified;
• 3) a PCR primer S-prim providing, in combination with the PCR primer as defined in 2), amplification of at least one amplicon comprised in said target sequence;
• 4) a donor oligonucleotide having the following general structure:
• wherein
• A′ represents a nucleic acid sequence motif of 10 to 30 bases being complementary to the nucleic acid sequence motif of A;
• 5) a restriction enzyme;
• and
• 6) amplification enzyme and nucleotides.