Nucleotide compounds including a rigid linker

• US 6,197,555 B1
• Filed: 12/14/1999
• Issued: 03/06/2001
• Est. Priority Date: 05/29/1998
• 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., Zhen, Weiguo
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/461,510
• Time in Patent Office: 448 Days
• Field of Search: 435/87, 435/88, 435/89, 435/91.1, 435/91.2, 536/22.1, 536/24.3, 536/25.3, 536/25.32, 536/25.33, 536/25.34, 536/26.21, 536/26.6, 536/26.8
• 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 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

A nucleoside/tide compound having the structure

NUC-L-S-LB/LG

is described wherein NUC is a nucleoside/tide having a nucleobase portion B, L is a rigid linkage, S is a spacer; and LB/LG is a member of a linkage pair or a label. NUC is attached to L through B such that when B is a purine, L is attached to the 8-position of the purine, when B is 7-deazapurine, L is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, L is attached to the 5-position of the pyrimidine. In an important aspect of the invention, L has the structure

wherein each of n, o and p are integers ranging from 0 to 3, and the sum of n, o and p is at least 2, and each of W, X, Y and Z is selected from the group consisting of carbon and nitrogen. The invention further includes polynucleotide compounds comprising the nucleoside/tide, and primer extension methods utilizing the nucleoside/tide, particularly when used in combination with certain mutant polymerase enzymes.

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 for forming a primer-template hybrid; and

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

2. The method of claim 1 wherein the primer extension reagent includes a theromostable DNA polymerase.

3. The method of claim 2 wherein the thermostable polymerase is a Taq polymerase.

4. The method of claim 3 wherein the Taq polymerase is a mutant Taq polymerase having a mutation at the F667 position.

5. The method of claim 4 wherein the mutation is F667Y.

6. The method of claim 3 wherein the Taq polymerase is a mutant Taq polymerase having a mutation at the R660 position.

7. The method of claim 3 wherein the mutation is selected from the group consisting of R660C, R660D and R660S.