Primers, probes and methods for nucleic acid amplification
First Claim
1. A method for analyzing 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 detecting double-stranded amplicons by homogeneous fluorescence detection, detecting said at least one single-stranded amplification product by sequence-specific homogeneous fluorescence detection at a temperature below the at least one primer-annealing temperature, and calculating the ratio fluorescence of said single-stranded 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.
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Citations
24 Claims
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1. A method for analyzing 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
detecting double-stranded amplicons by homogeneous fluorescence detection, detecting said at least one single-stranded amplification product by sequence-specific homogeneous fluorescence detection at a temperature below the at least one primer-annealing temperature, and calculating the ratio fluorescence of said single-stranded product to the fluorescence of said double-stranded amplicons.
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16. A double-stranded nucleic acid hybridization probe for a preselected nucleic acid target sequence comprising
a) a first oligonucleotide comprising a first sequence perfectly complementary to said target sequence and having a calculated melting temperature (Tm) with respect to said target sequence of 30-55° - C.;
b) a second oligonucleotide comprising a second sequence that is complementary to said first sequence but is shorter than said first sequence by up to ten nucleotides;
c) a fluorophore label attached to said first sequence that is excitable by fluorescence emission from a fluorescent DNA dye but is not excitable at the maximum absorption wavelength of said dye; and
d) a non-fluorescent quencher attached to said second sequence. - View Dependent Claims (17)
- C.;
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18. A low-temperature, mismatch tolerant, single-stranded oligonucleotide hybridization probe having a first calculated melting temperature with respect to its perfect complement of not more than 60°
- C., preferably not more than 50°
C., said probe including a stem-loop hairpin having a stem whose calculated melting temperature is at least 10°
C. lower than said first melting temperature but not lower than 30°
C., said probe including a fluorophore capable of absorption of fluorescence energy emitted by a fluorescent DNA dye and means to quench fluorescence that would result from exposing said probe to said dye and exciting the dye in the absence of a target for the probe.
- C., preferably not more than 50°
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19. A low-temperature linear oligonucleotide hybridization probe
a) that is mismatch tolerant at temperatures in the range of 30-60° - C.;
b) that forms secondary structure in the temperature range of 30°
C. to 50°
C.; and
c) is dual-labeled with (i) a fluorophore that is excitable by fluorescence emission from at least one fluorescent DNA dye but is not excitable at the wavelength appropriate for exciting said dye; and
(ii) a non-fluorescence quencher located so as to substantially quench fluorescence associated with said secondary structure but not florescence associated with a probe-target hybrid. - View Dependent Claims (20)
- C.;
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21. A sequential amplification-Pyrosequencing method comprising,
a) amplifying a DNA substrate by a LATE-PCR amplification reaction to prepare single stranded DNA product; -
b) removing pyrophosphate and, if present, leftover dNTPs;
c) if necessary, adding an oligonucleotide to block the excess primer d) adding Pyrosequencing reagents before adding sequencing primer if pyrophosphate and dNTPs have not already been removed as in b) above;
e) adding sequencing primer; and
f) performing Pyrosequencing. - View Dependent Claims (22, 23, 24)
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Specification