Specific nucleic acid amplification with compounded selectivity
The present disclosure provides, in various aspects and embodiments, methods and compositions for selectively amplifying a rare target nucleic acid and/or suppressing amplification of non-target nucleic acids with sequences similar to the rare target nucleic acid. The methods and composition are useful, for example, for detecting rare alleles among a population of wild-type alleles.
1. A composition, comprising:
a pseudo-target nucleic acid and/or a target nucleic acid that comprises a specificity domain binding site and a priming domain binding site that is downstream from the specificity domain binding site; and
an engineered primer that comprises (a) a specificity domain linked to a priming domain via a linker comprising a non-nucleotide moiety and (b) a competitive domain that binds to the specificity domain of the engineered primer or to the specificity domain binding site of the pseudo-target nucleic acid and/or a target nucleic acid,
wherein the specificity domain binds to the specificity domain binding site on the target nucleic acid and the priming domain binds to the priming domain binding site that is downstream from the specificity domain binding site on the target nucleic acid.
2. The composition of claim 1 further comprising a polymerase.
3. The composition of claim 2 , wherein the polymerase is a DNA polymerase or a reverse transcriptase.
4. The composition of claim 3 , wherein the polymerase is a DNA polymerase selected from Vent polymerase, Bsm polymerase, Bst polymerase, Csa polymerase, 96-7 polymerase, and Pfu polymerase.
5. The composition of claim 1 , wherein the engineered primer comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA) and/or a morpholino.
6. The composition of claim 1 , wherein the specificity domain and the priming domain are linked to each other through a 5′-3′ linkage or a 5′-5′ linkage.
7. The composition of claim 6 , wherein the specificity domain comprises a 3′ end having a blocking moiety.
8. The composition of claim 1 , wherein the priming domain is shorter than the specificity domain.
9. The composition of claim 1 , wherein the specificity domain and the competitive domain are covalently linked to each other to form a hairpin structure.
10. The composition of claim 1 , wherein the target nucleic acid comprises at least one mutation relative to its wild-type counterpart nucleic acid.
11. The composition of claim 1 , wherein the non-nucleotide moiety is a chemical moiety.
12. The composition of claim 11 , wherein the chemical moiety is selected from the group consisting of a polyethylene glycol linkage, an alkyl spacer, a PNA linkage or a LNA linkage.