Dye-labeled ribonucleotide triphosphates

• US 7,262,029 B2
• Filed: 12/08/2004
• Issued: 08/28/2007
• Est. Priority Date: 06/22/2001
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Khan, Shaheer H., Fisher, Peter Virgil, Vatta, Paolo
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/006,691
• Publication Number:
  US 20050100948A1
• Time in Patent Office: 993 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/91.1
• 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. 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.