Nucleotide compositions comprising photocleavable markers and methods of preparation thereof

• US 7,547,530 B2
• Filed: 02/09/2006
• Issued: 06/16/2009
• Est. Priority Date: 07/13/2001
• Status: Active Grant

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Olejnik, Jerzy, Rothschild, Kenneth J., Krzymanska-Olejnik, Edyta
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/351,996
• Publication Number:
  US 20060252923A1
• Time in Patent Office: 1,223 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/91.1
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0032 Methine dyes, e.g. cyanine dyes
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Labelled nucleotides and polynucleotides useful in the sequencing of nucleic acids are described. Methods of preparing photocleavable marker nucleotides and photocleavable marker-polynucleotide conjugates are described. Such photocleavable markere nucleotides can be incorporated into nucleic acid so as to create photocleavable marker-polynucleotide conjugates.

Claims

1. A conjugate, comprising a nucleotide attached to a fluorescent marker through a photocleavable linker, said marker comprising BODIPY.

2. The conjugate of claim 1, wherein said nucleotide is a ribonucleotide.

3. The conjugate of claim 1, wherein said nucleotide is a deoxyribonucleotide.

4. The conjugate of claim 1, wherein said cleavable linker is a photocleavable linker.

5. A method, comprising:

a) providing;

i) nucleic acid,

ii) the conjugate of claim 1, and

iii) a nucleic acid-modifying enzyme;

b) mixing said nucleic acid and said nucleic acid-modifying enzyme in the presence of said conjugate under conditions such that said conjugate is incorporated into said nucleic acid to produce labeled nucleic acid.

6. The method of claim 5, wherein said nucleic acid is RNA.

7. The method of claim 5, wherein said nucleic acid is DNA.

8. The method of claim 5, wherein said nucleic acid-modifying enzyme is a polymerase.

9. The method of claim 5, wherein said nucleic acid-modifying enzyme is a terminal transferase.

10. The method of claim 5, wherein said nucleic acid-modifying enzyme is a ligase.