Late-pcr
AssigneeBrandeis University
InventorsLawrence J. Wangh, Kenneth Pierce, Cristina Hartshorn, John Rice, J. Aquiles Sanchez
Life of this patent
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Abstract
A non-symmetric polymerise 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.
Claims38
-49. (canceled)
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,
- (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.
A thermal regulated device for carrying out linear amplification of at least one single-stranded DNA product and removal of said at least one single-stranded DNA product from the reaction via hybridization and subsequent harvesting of said at least one single-stranded DNA product.
A kit comprising:
- (a) at least one pair of PCR primers for a DNA target, 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;
- and
- (b) at least one capture probe for at least one single-stranded DNA product.
Dependencies are inferred from the claim text.