Universal reporter-based genotyping methods and materials
AssigneeLife Technologies Corporation
InventorsKelly Li, Shoulian Dong, Caifu Chen
Life of this patent
Estimating the term…
Abstract
The present disclosure is drawn to methods for detection, quantitation and analysis of nucleotides of interest, for example SNPs, in nucleic acid sequences of interest using universal FRET-based reporter primers.
Claims23
A method for determining the genotype of a target nucleic acid molecule, the method comprising:
- providing one or more target nucleic acid molecules and a first allele-specific primer, which is specific for a first allele, comprising a first 5′-universal tail which comprises a binding site for a first universal FRET-based reporter primer (UFP), wherein the first UFP comprises a first fluorophore and at least a first quencher moiety;
- and a second allele-specific primer which is specific for a second allele, comprising a second 5′-universal tail which comprises a binding site for a second UFP, wherein the second UFP comprises a second fluorophore and at least a second quencher moiety;
- wherein the first and second fluorophore are different and the first quencher moiety and the second quencher moiety are the same or different;
- providing a locus specific primer;
- amplifying the target nucleic acids;
- measuring the intensities of the first and the second fluorophore;
- and
- analyzing nucleic acid genotypes based on the intensities of the first and second fluorophores.
A method for determining the genotype of a target nucleic acid molecule, the method comprising:
- providing one or more target nucleic acid molecules and a first allele-specific primer, which is specific for a first allele, comprising a first 5′-universal tail which comprises a binding site for a first UFP, wherein the first UFP comprises a first fluorophore;
- and a second allele-specific primer which is specific for a second allele, comprising a second 5′-universal tail which comprises a binding site for a second UFP, wherein the second UFP comprises a second fluorophore;
- wherein the first and second fluorophore are different and are not directly excited by excitation light;
- providing a locus specific primer;
- providing a double-stranded nucleic acid intercalator dye;
- wherein the double-stranded nucleic acid intercalator dye is configured to be directly excited by the excitation light and its fluorescence excites the first and the second fluorophores;
- amplifying the target nucleic acids;
- measuring the intensities of the first and the second fluorophore;
- and
- analyzing nucleic acid genotypes based on the intensities of the first and second fluorophores.
A reaction mixture for analyzing, quantitating or detecting one or more alleles or polymorphisms in a target nucleic acid, wherein the reaction mixture comprises a first allele-specific primer, which is specific for a first allele, comprising a first 5′-universal tail which comprises a binding site for a first universal FRET-based reporter primer (UFP), wherein the first UFP comprises a first fluorophore and at least a first quencher moiety;
- and a second allele-specific primer which is specific for a second allele, comprising a second 5′-universal tail which comprises a binding site for a second UFP, wherein the second UFP comprises a second fluorophore and at least a second quencher moiety;
- wherein the first fluorophore and the second fluorophore are different and the first quencher moiety and the second quencher moiety are the same or different.
16-31. (canceled)
A kit for analyzing, quantitating or detecting one or more alleles or polymorphisms in a target nucleic acid, wherein the kit comprises a first allele-specific primer, which is specific for a first allele, comprising a first 5′-universal tail which comprises a binding site for a first universal FRET-based reporter primer (UFP), wherein the first UFP comprises a first fluorophore and at least a first quencher moiety;
- and a second allele-specific primer which is specific for a second allele, comprising a second 5′-universal tail which comprises a binding site for a second UFP, wherein the second UFP comprises a second fluorophore and at least a second quencher moiety;
- wherein the first and second fluorophore are different and the at least first quencher moiety and the at least second quencher moiety are the same or different.
(canceled)
(canceled)
Dependencies are inferred from the claim text.