Dye-labeled ribonucleotide triphosphates

• US 20050100948A1
• Filed: 12/08/2004
• Published: 05/12/2005
• Est. Priority Date: 06/22/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Khan, Shaheer H., Fisher, Peter Virgil, Vatta, Paolo
• Granted Patent: US 7,262,029 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

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.

Claims

1-117. -117. (canceled)

118. 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 and the dye-labeled rCTP compound is incorporated in the primer extension products;

hydrolyzing 3′

-5′

phosphodiester linkages between adjacent ribo- and deoxyribonucleotides to produce fragments;

resolving the fragments by size; and

detecting the fragments.

119. The method according to claim 118, wherein the template DNA is genomic DNA.

120. The method according to claim 118, wherein the fragments that contain primers are separated from other fragments before the fragments that contain primers are resolved by size.

121. The method according to claim 118, wherein at least one primer is biotinylated.

122. The method according to claim 118, wherein at least one primer is a hybridization based pull-out primer.

123. The method according to claim 118, wherein one primer comprises a modified base preventing primer extension in the 5′

direction.