A method for amplifying nucleic acid for differentiating a first nucleic acid sequence from a second nucleic acid sequence wherein the first and the second nucleic acid are in the same amplification reaction mixture, comprising:
- (a) providing or having provided a selector polynucleotide, wherein the selector polynucleotide contains therein a selector nucleotide residue,
- wherein the selector polynucleotide comprises at least a single residue selector nucleotide that is located at the first (ultimate), second (penultimate), third (antepenultimate), fourth (preantepenultimate), fifth (propreantepenultimate), or sixth (one residue to the 5′ side of the propreantepenultimate) position in reference to a 3′ end, or is at a position that is even more distal to the 3′ end,
- wherein the at least single residue selector nucleotide is structurally dissimilar to all other nucleotide residues within the region of the selector polynucleotide needed for annealing or hybridizing of the selector polynucleotide to a target nucleic acid, and
- the selector polynucleotide has a 3′ end that can be:
- extended by a DNA polymerase, or processed to have a 3′ end that can be extended by a DNA polymerase;
- (b) providing or having provided a DNA polynucleotide or plurality of DNA polynucleotides;
- (c) contacting, annealing or hybridizing, the selector polynucleotide to the DNA polynucleotide or plurality of DNA polynucleotides, wherein the DNA polynucleotide or plurality of DNA polynucleotides acts as a template DNA polynucleotide to the selector polynucleotide under conditions wherein the selector polynucleotide anneals or specifically hybridizes to a complementary sequence or substantially complementary sequence in the DNA polynucleotide or plurality of DNA polynucleotides, thereby generating a nucleic acid duplex;
- wherein the selector polynucleotide is either matched to the template DNA polynucleotide at the position of the selector nucleotide residue or is not matched to the template DNA polynucleotide at the position of the selector nucleotide residue;
- (d) contacting the nucleic acid duplex with a DNA polymerase enzyme having 5′ to 3′ extension activity and having a 3′ to 5′ exonuclease activity, and/or a DNA polymerase enzyme having 5′ to 3′ extension activity and an enzyme having 3′ to 5′ exonuclease activity, under conditions wherein the DNA polymerase enzyme and/or the polymerase enzyme having 5′ to 3′ extension activity and the enzyme having 3′ to 5′ exonuclease activity are enzymatically active,
- wherein either:
- (i) the selector nucleotide residue is mismatched between the selector polynucleotide and the DNA polynucleotide or plurality of DNA polynucleotides, and the 3′ to 5′ exonuclease activity results in enzymatically removing portions of the selector polynucleotide from the 3′ end including the selector nucleotide residue and all nucleotides 3′ of the selector nucleotide prior to the DNA polymerase enzymatically extending what remains of the selector polynucleotide to generate a new extended DNA polynucleotide that does not retain the selector nucleotide residue);
- or
- (ii) the DNA polymerase enzymatically extends a selector polynucleotide that is matched at the selector nucleotide residue with the DNA polynucleotide or plurality of DNA polynucleotides, without removing the selector nucleotide, thus retaining or incorporating the selector nucleotide residue of the selector polynucleotide into a new extended DNA polynucleotide;
- and
- (e) contacting the nucleic acid duplex with a ribonuclease enzyme,
- wherein:
- (i) if the selector nucleotide residue has been removed by the 3′ to 5′ exonuclease activity, then the portion of the selector polynucleotide that was 5′ to the single selector nucleotide is retained within the new extended synthetic polynucleotide;
- or
- (ii) if the selector nucleotide residue is retained in the extended selector polynucleotide and is matched to a deoxyribonucleotide residue, then the ribonuclease enzyme cuts at the selector nucleotide residue thereby detaching the portion of the new extended polynucleotide that was 5′ to the selector nucleotide residue,
- wherein a new extended polynucleotide that retains the portion of the selector polynucleotide that had been 5′ to the selector nucleotide residue is exponentially amplified,
- and wherein a new extended polynucleotide that has had the portion of the selector polynucleotide that was 5′ to the selector nucleotide residue detached from the extended polynucleotide cannot be exponentially amplified and amplification is thereby selectively suppressed, thereby differentiating the first nucleic acid sequence from the second nucleic acid sequence.