Suppression of non-specific amplification with high-homology oligonucleotides
The invention comprises suppressor oligonucleotides for reducing amplification of a non-target nucleic acid sequences; the method of designing and using such oligonucleotides, as well as kits and reaction mixtures.
1. A reaction mixture for performing an amplification reaction of a target sequence in the genome of a target organism with reduced amplification of non-target sequences, comprising:
at least one allele-specific primer specific for a variant of the target sequence;
an amplicon formed by the at least on allele-specific primer and a reverse primer;
a suppressor oligonucleotide (a) having no significant similarity with the target sequence as determined by the blastn algorithm for somewhat similar sequences, (b) extendable by a DNA polymerase, and (c) having a sequence comprising at least one region of complementarity to multiple sites in the genome of the target organism, wherein the least one region of complementarity: (i) is at least 15 base pairs long; and (ii) spans the 3′-end of the suppressor oligonucleotide; and
a fluorescently labeled probe oligonucleotide, wherein the reaction mixture does not include an amplicon formed by the suppressor oligonucleotide.
2. The reaction mixture of claim 1 , wherein the suppressor oligonucleotide is selected from a group consisting of SEQ ID NOs: 1-3, and 5.
3. A reaction mixture for performing an amplification reaction of a target sequence in the genome of a target organism with reduced amplification of non-target sequences, comprising:
at least one allele-specific primer specific for a variant of the target sequence comprising at least one unconventional-base nucleotide;
an amplicon formed by the at least one allele-specific primer and a reverse primer;
a suppressor oligonucleotide (a) having no significant similarity with the target sequence as determined by the blastn algorithm for somewhat similar sequences, (b) extendable by a DNA polymerase, and (c) having a sequence comprising at least one region of complementarity to multiple sites in the genome of the target organism, wherein the least one region of complementarity: (i) is at least 15 base pairs long; and (ii) spans the 3′-end of the suppressor oligonucleotide;
buffer necessary for the function of a DNA polymerase; wherein the reaction mixture does not include an amplicon formed by the suppressor oligonucleotide.
4. The reaction mixture of claim 3 , wherein the suppressor oligonucleotide is selected from a group consisting of SEQ ID NOs: 1-3, and 5.
5. The reaction mixture of claim 3 , wherein said unconventional-base nucleotide is alkylated.
6. The reaction mixture of claim 1 , further comprising nucleotide triphosphates, a DNA polymerase, and a buffer necessary for the function of the polymerase.
7. The reaction mixture of claim 1 , further comprising nucleotide triphosphates, a DNA polymerase, and a buffer necessary for the function of the polymerase.