Propargylethoxyamino nucleotide primer extensions

• US 6,248,568 B1
• Filed: 06/18/1998
• Issued: 06/19/2001
• Est. Priority Date: 08/12/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: The Perkin-Elmer Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/583,068
• Time in Patent Office: 1,097 Days
• Field of Search: 435/89, 435/90, 435/91.1, 435/6, 536/24.33
• US Class Current: 435/91.1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

Propargylethoxyamino nucleosides are disclosed having the structure

wherein R₁ and R₂ are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W₁ is —H or —OH; W₂ is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W₃ is —PO₄, —P₂O₇, —P₃O₁₀, phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Claims

1. A method for performing a primer extension reaction comprising the steps of:

providing a template nucleic acid;

annealing an oligonucleotide primer to a portion of the template nucleic acid; and

adding primer-extension reagents to the primer-template hybrid for extending the primer, the primer extension reagents including a nucleoside compound having the structure;

 

wherein;

R₁ and R₂ taken separately are selected from the group consisting of —

H, lower alkyl, protecting group, and label;

B is a 7-deazapurine, purine, or pyrimidine nucleoside base;

wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N⁹-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N¹-position of the pyrimidine; and

wherein if B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine if B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and if B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;

W₁ is selected from the group consisting of —

H and —

OH;

W₂ is —

OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′

-position; and

W₃ is selected from the group consisting of —

PO₄, —

P₂O₇, —

P₃O₁₀, phosphate analog, and —

OH.

2. The method of claim 1 wherein one of R₁ and R₂ is label and the other is —

H.