Modified oligonucleotides for mismatch discrimination

• US 6,949,367 B1
• Filed: 11/28/2000
• Issued: 09/27/2005
• Est. Priority Date: 04/03/1998
• Status: Expired due to Term

• Current Assignee: ELITechGroup Inc. (Bruker Corporation)
• Original Assignee: Epoch Pharmaceuticals, Inc.
• Inventors: Dempcy, Robert O., Kutyavin, Igor V., Singer, Michael J., Afonina, Irina A.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US09/724,959
• Time in Patent Office: 1,764 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/22.1, 536/24.32, 935/6, 935/77, 935/78, 436/518
• US Class Current: 435/91.1
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical

Abstract

Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Claims

1. A method for primer extension, the method comprising incubating a polynucleotide containing a target sequence with one or more oligonucleotide primers complementary to the target sequence, in the presence of a polymerizing enzyme and nucleotide substrates under conditions favorable for polymerization;

wherein at least one of the oligonucleotide primers contains a modified base selected from the group consisting of an unsubstituted pyrazolo[3,4-d]pyrimidine, a 3-substituted pyrazolo[3,4-d]pyrimidine and a 5-substituted pyrimidine base, in place of a purine or pyrimidine base.

2. A method in accordance with claim 1, wherein one of said oligonucleotide primers is extended with a single base.

3. A method in accordance with claim 1, wherein said at least one of said oligonucleotide primers further comprises an attached minor groove binder.

4. A method in accordance with claim 1, wherein said incubating is part of an amplification reaction.

5. A method in accordance with claim 4, wherein said amplification reaction is a polymerase chain reaction.

6. A method in accordance with claim 1, wherein said modified oligonucleotide further comprises a covalently attached minor groove binder.

7. A method for determining the nucleotide sequence of a polynucleotide, the method comprising:

(a) incubating the polynucleotide with a modified oligonucleotide array under hybridization conditions; and

(b) determining to which of the modified oligonucleotides in the array the polynucleotide hybridizes;

wherein a plurality of the modified oligonucleotides comprise at least one 3-substituted pyrazolo[3,4]pyrimidine in place of a purine base.

8. A method in accordance with claim 7, wherein said array comprises from 10 to 100,000 different modified oligonucleotides.

9. A method in accordance with claim 7, wherein said array comprises from 10 to 1000 different modified oligonucleotides.