Dye-labeled ribonucleotide triphosphates
View Patent ↗The invention provides novel dye-labeled ribonucleotide analogs and methods for synthesizing those analogs. The compounds of the invention are especially useful for DNA sequencing by the polymerase chain reaction.
1. A method for detecting 5-methylcytosine in a DNA template comprising:
treating the DNA template with a bisulfite salt under conditions whereby 5-methylcytosine remains non-reactive;
incubating the DNA template with a DNA polymerase, dATP, dGTP, dCTP, dTTP, at least two oligonucleotide primers complementary to the DNA template, and a dye-labeled rCTP compound so that the primers are extended to form primer extension products and the dye-labeled rCTP compound is incorporated in the primer extension products;
hydrolyzing 3′-5′ phosphodiester linkages between adjacent ribo- and deoxyribonucleotides in the primer extension products to produce fragments;
resolving the fragments by size; and
detecting the fragments in order to detect 5-methylcytosine in the DNA template.
2. The method according to claim 1 , wherein the template DNA is genomic DNA.
3. The method according to claim 1 , wherein the fragments that contain primers are separated from other fragments before the fragments that contain primers are resolved by size.
4. The method according to claim 1 , wherein at least one primer is biotinylated.
5. The method according to claim 1 , wherein at least one primer is a hybridization based pull-out primer.
6. The method according to claim 1 , wherein one primer comprises a modified base preventing primer extension in the 5′ direction.