Allele specific primer extension

• US 20030104372A1
• Filed: 02/23/2001
• Published: 06/05/2003
• Est. Priority Date: 12/23/1996
• Status: Abandoned Application

• Current Assignee: Pyrosequencing AB
• Original Assignee: Pyrosequencing AB
• Inventors: Nyren, Pal, Lundeberg, Joakim, Ahmadian, Afshin
• Application Number:
  US09/791,190
• Publication Number:
  US 20030104372A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

The present invention provides methods of allele-specific primer extension useful for detecting mutations and genetic variations.

Claims

1. A method for detecting an allele-specific base at a predetermined position in a target nucleic acid molecule comprising providing a first and a second hybridization mixture, said first hybridization mixture comprising said target nucleic acid molecule, and a primer that hybridizes to a region of said target nucleic acid molecule and that has a 3′

-terminus that is complementary to a non-mutated base at said predetermined position, said second hybridization mixture comprising said target nucleic acid molecule, and a primer that hybridizes to a region of said target nucleic acid molecule and that has a 3′

-terminus that is complementary to a mutated base at said predetermined position;

adding a primer extension reaction mixture to each of said first and second hybridization mixtures, said primer extension reaction mixture comprising a DNA polymerase, nucleotides, and a nucleotide degrading enzyme; and

determining primer extension efficiency in each of said first and second mixtures, whereby greater efficiency in the mixture comprising the primer having a 3′

-terminus that is complementary to the mutated base is indicative of the presence of the mutated base in the target nucleic acid, and whereby greater efficiency in the mixture comprising the primer having a 3′

-terminus that is complementary to the non-mutated base is indicative of the presence of the non-mutated base in the target nucleic acid.

2. The method of claim 1 wherein the nucleotide degrading enzyme is apyrase.

3. The method of claim 1 wherein the target nucleic acid is amplified before hybridization.

4. The method of claim 1 wherein the target nucleic acid is immobilized.

5. The method of claim 1 wherein primer extension efficiency is measured by an assay selected from mass spectroscopy, a luminometric assay, a fluorescent assay, and pyrosequencing.

6. The method of claim 1 wherein said method is performed in a solid phase microarray format.

7. The method of claim 1 wherein said primers are tagged on the 5′

-ends by barcodes.

8. The method of claim 1 wherein the target nucleic acid is double stranded.

9. A method for detecting an allele-specific base at a predetermined position in a target nucleic acid comprising conducting a first and a second allele-specific primer extension reaction using a first and a second primer, respectively that hybridizes to a region of said target nucleic acid, each of said primers having:

1) a 3′

-end base that is complementary to the base that is 5′

of the predetermined position in the target;

     2) a base one position from the 3′

-end that in the first primer is complementary to a non-mutated based at the predetermined position, and in the second primer is complementary to a mutated base at the predetermined position; and

     3) a base two positions from the 3′

-end that is the same as the base that is 3′

of the predetermined position in the target; and

determining primer extension efficiency in said first and second reactions, whereby greater efficiency in said first reaction is indicative of the presence of a non-mutated base at said predetermined position, and whereby greater efficiency in said second reaction is indicative of the presence of a mutated base at said predetermined position.

10. The method of claim 9 wherein said extension reactions are performed in the presence of a nucleotide degrading enzyme.

11. The method of claim 10 wherein said nucleotide degrading enzyme is apyrase.

12. The method of claim 9 wherein said target nucleic acid is amplified before hybridization.

13. The method of claim 9 wherein said target nucleic acid is immobilized.

14. The method of claim 9 wherein said primer extension efficiency is measured by an assay selected from mass spectroscopy, a luminometric assay, a fluorescent assay, and pyrosequencing.

15. The method of claim 9 wherein said method is performed in a solid phase microarray format.

16. The method of claim 9 wherein said primers are tagged at the 5′

-ends by barcodes.

17. The method of claim 9 wherein said target nucleic acid is double stranded.