Patent ID: 8071338
Filing Date: 2011-12-06
Classification: C12Q

Abstract:
1. A method of detecting a target sequence in a polynucleotide in a biological sample, wherein the sample also contains a second polynucleotide comprising a second sequence, wherein the second sequence differs from the target sequence by at least one nucleotide, the method comprising, i. contacting the sample with a blocker oligonucleotide under conditions to allow for hybridization of the blocker oligonucleotide to the second sequence or the target sequence, wherein the blocker oligonucleotide comprises a non-natural nucleotide that results in a higher melting temperature of the blocker oligonucleotide compared to insertion of a naturally-occurring nucleotide in the non-natural nucleotide's position; ii. contacting the sample in the presence of the hybridized blocker oligonucleotide with at least one primer and a polymerase significantly lacking 5′-3′ nuclease activity under conditions such that template-dependent extension of the primer occurs, wherein the primer hybridizes to the polynucleotides, upstream of the blocker oligonucleotide-hybridizing sequence; wherein the blocker oligonucleotide hybridizes to the second sequence sufficiently to impair amplification of the second sequence by the polymerase, wherein the blocker oligonucleotide does not comprise an intercalating nucleotide, and further wherein hybridization of the oligonucleotide to the target sequence does not significantly impair amplification of the target sequence by the polymerase significantly lacking 5′-3′ nuclease activity, wherein the blocker oligonucleotide hybridized to the second sequence would not block a polymerase having 5′-3′ nuclease activity.