Fluorescence polarization in nucleic acid analysis

A method of diagnosing the presence of a genetic polymorphism characterized by the presence of a single nucleotide target site in a subject, comprising:

• (a) isolating genomic DNA or RNA from a sample of biological material, wherein template genomic DNA or template DNA prepared from the RNA comprises one or more target sites, and wherein each target site comprises a genetic polymorphism;
• (b) forming a fluorescently labeled oligonucleotide bound to the target site if present wherein the oligonucleotide comprises a fluorophore linked to a terminator contained therein;
• (c) detecting fluorescence polarization of the fluorophore of the fluorescently-labeled oligonucleotide;
• wherein the oligonucleotide is formed from a primer bound to the template immediately 3′ to the target site and a terminator covalently linked to a fluorophore, and wherein said terminator-linked fluorophore binds to the target side and reacts with the primer to produce an extended primer which is said fluorescently labeled oligonucleotide,
• wherein an increase in fluorescence polarization indicates the presence of the target site
• thereby detecting the presence of the target site which comprises a genetic polymorphism by said increase in fluorescence polarization.

A method for detecting the presence of specified nucleotides at a first and second target single nucleotide site in each of two alleles in templates which comprise amplified genomic DNA which method comprises

• (a) forming a first fluorescently labeled oligonucleotide from a first primer bound to a first template allele immediately 3′ to the target site and a first terminator covalently linked to a first fluorophore, and wherein said terminator linked fluorophore binds to a first target site if present and reacts with the primer to produce an extended primer which is said first fluorescently labeled oligonucleotide, and
• forming a second fluorescently labeled oligonucleotide from said primer bound to a second template allele immediately 3′ to the target site and a second terminator covalently linked to a second fluorophore, and wherein said terminator linked fluorophore binds to a second target site if present and reacts with the primer to produce an extended primer which is said second fluorescently labeled oligonucleotide, and
• (b) detecting fluorescence polarization of the fluorophores of each of said first and second differently fluorescently labeled oligonucleotides
• whereby an increase in fluorescence polarization of said first fluorophore indicates the presence of the first target site, and
• whereby an increase in fluorescence polarization of said second fluorophore indicates the presence of the second target site.

A method for detecting the presence of a target site of at least one nucleotide in a template nucleic acid comprising detecting fluorescence polarization of a fluorescently labeled oligonucleotide which is bound to the target site

• wherein an increase in fluorescence polarization indicates the presence of the target site, thereby detecting the presence of the target site by said increase in fluorescence polarization;
• and
• wherein the fluorescently labeled oligonucleotide comprises a fluorophore linked to a terminator therein;
• and wherein the fluorescently labeled oligonucleotide has been formed from a primer bound to the template immediately 3′ to the target site and terminator covalently linked to a fluorophore, and wherein said terminator-linked fluorophore binds to the target site and reacts with the primer to produce an extended primer which is said fluorescently labeled oligonucleotide.