Late-PCR
First Claim
1. A method for amplification of at least one single-stranded DNA product via a non-symmetric polymerase chain reaction (PCR) comprising:
- a) thermally cycling a PCR reaction mixture containing at least one DNA or cDNA target, at least one pair of PCR primers for said target, dNTP'"'"'s, and a thermostable DNA polymerase through repeated cycles of strand melting, primer annealing, and primer extension, wherein;
(i) the at least one PCR primer pair comprises a Limiting Primer and an Excess Primer,(ii) the Limiting Primer of said pair is present at a concentration of up to 200 nM, and the Excess Primer of said pair is present at a concentration at least five times higher than the Limiting Primer,(iii) an initial, concentration-adjusted melting temperature of the Limiting Primer of said pair that is equal to or greater than an initial, concentration-adjusted melting temperature of the Excess Primer of said pair, when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition,(iv) thermal cycling is repeated a number of times following exhaustion of the Limiting Primer sufficient for linear amplification of at least one single-stranded DNA product which is the extension product of the Excess Primer of said at least one pair of PCR primers; and
b) removing said at least one single-stranded DNA product from the reaction mixture by;
(i) hybridizing said product to a capture probe, or(ii) permitting product evolution via strand-to-strand hybridization and extension of said at least one single-stranded DNA product in the reaction, or(iii) converting at least a portion of said single-stranded DNA product in the reaction into a double-strand molecule via hybridization and extension of a Low-Tm or Super-Low Tm primer.
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Accused Products
Abstract
A non-symmetric polymerize chain reaction (PCR) amplification method employing a limiting primer in low concentration whose concentration-adjusted melting point at least equals, and preferably exceeds, that of the excess primer, the latter in turn not being more than 25° C. below the melting temperature of the amplicon. Assays employing such amplification and labeled hybridization probes, including assays that include a detection step following primer extension or a low-temperature probe, or both. Kits for performing such assays and primer or primer-and-probe sets for performing the foregoing amplifications and assays.
12 Citations
27 Claims
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1. A method for amplification of at least one single-stranded DNA product via a non-symmetric polymerase chain reaction (PCR) comprising:
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a) thermally cycling a PCR reaction mixture containing at least one DNA or cDNA target, at least one pair of PCR primers for said target, dNTP'"'"'s, and a thermostable DNA polymerase through repeated cycles of strand melting, primer annealing, and primer extension, wherein; (i) the at least one PCR primer pair comprises a Limiting Primer and an Excess Primer, (ii) the Limiting Primer of said pair is present at a concentration of up to 200 nM, and the Excess Primer of said pair is present at a concentration at least five times higher than the Limiting Primer, (iii) an initial, concentration-adjusted melting temperature of the Limiting Primer of said pair that is equal to or greater than an initial, concentration-adjusted melting temperature of the Excess Primer of said pair, when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (iv) thermal cycling is repeated a number of times following exhaustion of the Limiting Primer sufficient for linear amplification of at least one single-stranded DNA product which is the extension product of the Excess Primer of said at least one pair of PCR primers; and b) removing said at least one single-stranded DNA product from the reaction mixture by; (i) hybridizing said product to a capture probe, or (ii) permitting product evolution via strand-to-strand hybridization and extension of said at least one single-stranded DNA product in the reaction, or (iii) converting at least a portion of said single-stranded DNA product in the reaction into a double-strand molecule via hybridization and extension of a Low-Tm or Super-Low Tm primer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification