Allele-specific amplification of nucleic acids using blocking oligonucleotides for wild type suppression
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United States of America Patent
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Mar 6, 2018
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Dec 17, 2014
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Dec 17, 2014
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Abstract
A method is provided for allele-specific amplification, utilizing a blocking oligonucleotide including at least one nucleotide with a base covalently modified at the exocyclic amino group, the blocking oligonucleotide being perfectly complementary to a wild type (WT) sequence when hybridized forming a first complex having a first melting temperature (Tm), the blocking oligonucleotide being partially non-complementary, at one or more nucleotides, to a target mutant (MT) sequence when hybridized forming a second complex having a second melting temperature (Tm), wherein the first Tm is higher than the second Tm and having at least one nucleotide with a base covalently modified at the exocyclic amino group, wherein the blocking oligonucleotide becomes unhybridized from the target MT sequence during amplification but remains hybridized with the WT sequence inhibiting amplification of the WT sequence utilizing a polymerase lacking 5′-3′ nuclease activity.
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