Allele-specific mutation detection assay using an enzyme-disabling agent

• US 7,262,032 B2
• Filed: 10/21/2005
• Issued: 08/28/2007
• Est. Priority Date: 04/24/2003
• Status: Expired due to Fees

• Current Assignee: Afshin Ahmadian, Joakim Lundeberg
• Original Assignee: Afshin Ahmadian, Joakim Lundeberg
• Inventors: Lundeberg, Joakim, Ahmadian, Afshin
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Baughman; Molly E.
• Application Number:
  US11/256,694
• Publication Number:
  US 20060088872A1
• Time in Patent Office: 676 Days
• Field of Search: 435/6, 435/91.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/537 Protease
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention relates to a method of detecting a base at a pre-determined position in a nucleic acid molecule by performing enzymatic detection reactions using base-specific detection oligomers, where each oligomer is specific for a particular base at the predetermined position, and then comparing the enzymatic detection reactions to determine which base is present at the position, with an enzyme-disabling agent being present during the enzymatic detection reaction. In preferred embodiments the enzymatic detection reaction is an oligomer elongation extension reaction, catalysed by, among others, polymerase or ligase. Also disclosed are methods of performing the assay in a liquid phase and on microarrays.

Claims

1. A method of detecting a base at a pre-determined position in a nucleic acid molecule, which comprises performing enzymatic oligomer elongation reactions using base-specific detection oligomers, with each oligomer being specific for a particular base at the predetermined position, and comparing the enzymatic oligomer elongation reactions to determine which base is present at the position, wherein a protein degrading enzyme is present during the enzymatic oligomer elongation reactions at a concentration such that mismatched enzymatic elongation reactions are eliminated or reduced.

2. The method of claim 1 wherein the protein degrading enzyme is Proteinase K.

3. The method of claim 1 wherein the base-specific oligomers have a base-specific position at the 3'"'"' end, the 3'"'"'-1 position or the 3'"'"'-2 position.

4. The method of claim 1 wherein the base-specific oligomers are tagged.

5. The method of claim 4 wherein the tag is a barcode.

6. The method of claim 1 wherein the base-specific oligomers are labelled, directly or indirectly.

7. The method of claim 1 wherein the method is performed in a solid phase microarray format.

8. The method of claim 7 wherein the target nucleic acid molecule or base-specific oligomers are hybridized to the microarray after the enzymatic oligomer elongation reaction.

9. The method of claim 8 wherein the hybridization occurs to immobilized complementary oligonucleotides on the microarray.

10. The method of claim 7 wherein the base-specific oligomers or the nucleic acid molecule are immobilized in the array prior to the enzymatic oligomer elongation reactions.

11. The method of claim 7 wherein multiple enzymatic oligomer elongation reactions are carried out simultaneously or in parallel.

12. The method of claim 1 wherein the base-specific oligomers are annealed to the nucleic acid molecules under competitive conditions wherein the base-specific oligomers are competing to anneal to the nucleic acid molecule.

13. The method of claim 12 wherein the annealing step is performed at 50-95°

C. followed by cooling to 15-50°

C.

14. The method of claim 1 wherein the enzymatic elongation reactions are compared by determining the efficiency of the enzymatic oligomer elongation reaction for each of the base-specific oligomers and comparing them.

15. The method of claim 1 wherein the enzymatic oligomer elongation reactions are catalyzed by a polymerase enzyme.

16. The method of claim 15 wherein the base-specific oligomers are primers, and the primers are elongated via the addition of nucleotides.

17. The method of claim 16 wherein the nucleotides are labelled or are intermediary nucleotides to which a label can be attached.

18. The method of claim 16 wherein primer elongation is determined by detecting or monitoring pyrophosphate release.

19. The method of claim 16 wherein a fluorescently labelled probe is hybridized to the nucleic acid molecule prior to elongation of the primers, and elongation is detected via an increase in fluorescence level.

20. The method of claim 1 wherein the enzymatic oligomer elongation reactions are catalyzed by a ligase enzyme.

21. The method of claim 20 wherein the base-specific oligomers are padlock probes.

22. The method of claim 20 wherein the base-specific oligomers are probes, and a second probe is provided which hybridizes to the nucleic acid molecule next to the base-specific oligomers.

23. The method of claim 22 wherein the junction between the base specific probes and second probe is at the pre-determined position.

24. The method of claim 22 wherein the enzymatic ligation detection reactions are determined via detecting the size of the oligomer.

25. The method of claim 22 wherein the base-specific probes are tagged with a label and the second probe is tagged with a hook.

26. The method of claim 1 wherein 2, 3 or 4 base specific oligomers are used.

27. The method of claim 26 wherein 4 base specific oligomers are used to detect an unknown base at the pre-determined position.