Single nucleotide polymorphism analysis of highly polymorphic target sequences

• US 7,348,146 B2
• Filed: 09/29/2004
• Issued: 03/25/2008
• Est. Priority Date: 10/02/2003
• Status: Active Grant

• Current Assignee: Elitechgroup MDX LLC
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Dempcy, Robert O., Belousov, Yevgeniy, Vorobiev, Alexei, Lokhov, Sergey G.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/954,955
• Publication Number:
  US 20050118623A1
• Time in Patent Office: 1,273 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

Methods and probes are provided for the analysis of target sequences having two or more polymorphisms wherein one of the polymorphisms is to be distinguished and another polymorphism is to be masked.

Claims

1. A method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence having at least two single nucleotide polymorphisms wherein a first single nucleotide polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said polymorphisms being in a probe region of said target nucleic acid, the method comprising:

(a) combining a sample containing said target nucleic acid with one or more oligonucleotide primers adjacent to or overlapping with said probe region of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula;

2. The method of claim 1, wherein the minor groove binder (M) is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs.

3. The method of claim 1, wherein the minor groove binder (M) is a substituted dihydrocyclopyrroloindole triamide (DPI₃).

4. The method of claim 1, wherein the minor groove binder (M) is attacked to the 5′

end of the oligonucleotide conjugate.

5. The method of claim 1, wherein the minor groove binder (M) is attached to the 3′

end of the oligonucleotide conjugate.

6. The method of claim 1, wherein Q is a quencher with an absorption spectra between about 400 to 800 nm, and Fl is a fluorophore with emission wavelengths between about 400 to 800 nm.

7. The method of claim 1, wherein said fluorophore (Fl) is selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

8. The method of claim 1, wherein said quencher (Q) is selected from the group consisting of mono azo and bis azo dyes.

9. The method of claim 1, wherein said sugar phosphate backbone (A) comprises 8-25 nucleotides in length.

10. The method of claim 1, wherein the fluorescence is produced directly upon hybridization of the probe with said target nucleic acid sequence.

11. The method of claim 1, wherein the fluorescence is produced upon enzymatic cleavage of the quencher or the fluorophore from the conjugate probe upon hybridization with a target nucleic acid sequence.

12. The method of claim 1, wherein the universal base is selected from the group provided in Table 1.

13. The method of claim 1, wherein the promiscuous base is selected from the group provided in Table 2.

14. The method of claim 1, wherein the promiscuous (indiscriminative) base has the formula: