Base analogues

• US 6,444,682 B1
• Filed: 04/18/2000
• Issued: 09/03/2002
• Est. Priority Date: 07/31/1997
• Status: Expired due to Fees

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Nycomed Amersham Inc.
• Inventors: McDougall, Mark, Smith, Clifford, Hill, Fergal, Brown, Daniel, Kumar, Shiv, Nampalli, Satyam, Simmonds, Adrian Christopher, Loakes, David, Hamilton, Alan
• Primary Examiner(s): Shah, Mukund J.
• Assistant Examiner(s): Patel, Sudhaker B.
• Application Number:
  US09/463,501
• Time in Patent Office: 868 Days
• Field of Search: 544/299, 544/301, 544/302, 544/304, 536/22.1, 514/274, 514/269, 514/470, 514/471, 514/414, 514/23
• US Class Current: 514/274
• CPC Class Codes: A61P 31/12 Antivirals
• 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: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Nucleotide or base analogues having structure (3) or (4)

wherein X═O or NH or S and

each R⁶ is independently H or alkyl or alkenyl or alkoxy or aryl or a reporter moiety.

Claims

1. A compound having the structure selected from the group consisting of

where X═

O or NH or S

each R⁶ is independently H or alkyl or alkenyl or alkoxy or aryl or a reporter moiety,

provided that

in structure (3), when X is O, then at least one R⁶ is a reporter moiety which is a reactive group or signal moiety or solid surface joined to the remainder of the molecule by a linker of at least 3 chain atoms,

in structure (3), when X is NH, then at least one R⁶ is a reporter moiety,

and wherein the compound is a nucleoside or nucleoside analogue wherein Q is

 

where Z is O,

R¹, R², R³ and R⁴ are the same or different and each is H or OH

R⁵ is mono-, di-, or tri-phosphate,

or one of R² and R⁵ is a phosphoramidite or other group for incorporation in a polynucleotide chain.

2. The compound of claim 1, wherein a reporter moiety is present.

3. The compound of claim 2, wherein the reporter moiety is a signal moiety.

4. The compound of claim 2, wherein the reporter moiety is a reactive group or signal moiety or solid surface joined to the remainder of the molecule by a linker of at least 3 chain atoms.

5. The compound of claim 1, wherein R⁵ is triphosphate.

6. The compound of claim 1, wherein one of R² and R⁵ is selected from phosphoramidite and H-phosphonate.

7. A polynucleotide comprising at least one residue of the compound of claim 1.

8. The polynucleotide of claim 7, wherein the polynucleotide is DNA or RNA.

9. A chain extension method which comprises reacting a polynucleotide with a nucleoside triphosphate analogue of claim 1 in the presence of a polymerase or a terminal deoxynucleotidyl transferase enzyme.

10. A method of detecting the polynucleotide of claim 7, which method comprises using for detection an antibody which binds to a base component selected from the group consisting of structure (3) and structure (4).

11. A method of making cDNA which comprises incubating an RNA template with a monomer mixture including a nucleotide analogue as claimed in claim 1 in the presence of a reverse transcriptase.

12. A method of amplifying a polynucleotide by PCR which method comprises using a monomer mixture including a nucleotide analogue as claimed in claim 1.

13. A method of reducing compression artefacts in nucleic acid sequencing, comprising

(a) incorporating at least one nucleoside analogue of claim 1 into polynucleotides using a polymerase; and

(b) separating polynucleotides created by the polymerase by electrophoresis, wherein incorporation of the nucleoside analogue into one or more polynucleotides reduces detection of compression artefacts.

14. A method of detecting a residue of the compound of claim 1, the method comprising analyzing by mass spectrometry.