Primers, probes and methods for nucleic acid amplification
First Claim
1. A homogeneous detection method for at least one single-stranded amplification product of a non-symmetric nucleic acid amplification process that generates both double-stranded and single-stranded amplicons by extension of oligonucleotide primers by a DNA polymerase and that includes at least one primer-annealing temperature, comprising(a) forming an amplification reaction mixture that includes at least one nucleic acid target sequence and detection reagents,(b) amplifying said nucleic acid target sequence by said non-symmetric nucleic acid amplification process,(c) detecting double-stranded amplicons by a fluorescent signal,(d) detecting said at least one single-stranded amplification product by a sequence-specific fluorescent signal at a temperature below the at least one primer-annealing temperature, and(e) normalizing the sequence-specific fluorescent signal as a calculated ratio of fluorescence of said single-stranded amplification product to the fluorescence of said double-stranded amplicons.
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Accused Products
Abstract
Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.
30 Citations
12 Claims
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1. A homogeneous detection method for at least one single-stranded amplification product of a non-symmetric nucleic acid amplification process that generates both double-stranded and single-stranded amplicons by extension of oligonucleotide primers by a DNA polymerase and that includes at least one primer-annealing temperature, comprising
(a) forming an amplification reaction mixture that includes at least one nucleic acid target sequence and detection reagents, (b) amplifying said nucleic acid target sequence by said non-symmetric nucleic acid amplification process, (c) detecting double-stranded amplicons by a fluorescent signal, (d) detecting said at least one single-stranded amplification product by a sequence-specific fluorescent signal at a temperature below the at least one primer-annealing temperature, and (e) normalizing the sequence-specific fluorescent signal as a calculated ratio of fluorescence of said single-stranded amplification product to the fluorescence of said double-stranded amplicons.
Specification