Degenerate nucleobase analogs

• US 9,677,123 B2
• Filed: 03/07/2007
• Issued: 06/13/2017
• Est. Priority Date: 03/15/2006
• Status: Active Grant

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Inventors: Battersby, Thomas R.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US12/282,180
• Publication Number:
  US 20100055798A1
• Time in Patent Office: 3,751 Days
• Field of Search: 536 221, 435 6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to novel degenerate nucleobase analogs and degenerate nucleobase oligomers derived therefrom, and methods of using such degenerate nucleobase oligomers.

Claims

1. A polynucleotide duplex comprising a degenerate nucleobase oligomer hybridized to a corresponding polynucleotide template,

wherein the corresponding polynucleotide template comprises a first polymorphic site characterized by multiple natural nucleobase variations and a second polymorphic site characterized by multiple nucleobase variations;

and

wherein the degenerate nucleobase oligomer has a first polymorphic site characterized by multiple nucleobase variations and a second polymorphic site characterized by multiple nucleobase variations, wherein the oligomer comprises

a degenerate nucleobase complementary to multiple natural nucleobase variations at its first polymorphic site, wherein the degenerate nucleobase is

a pyrimidine or an analog thereof which is complementary to guanine or adenine and has hydrogen-bonding functionality arrayed such that the pyrimidine or analog thereof can form two hydrogen bonds in a wobble conformation with one natural purine and two hydrogen bonds in a reverse wobble conformation with the other natural purine;

or

a purine or an analog thereof which is complementary to cytosine, uracil or thymine and has hydrogen-bonding functionality arrayed such that the purine or analog thereof can form two hydrogen bonds in a wobble conformation with one pyrimidine-like component and two hydrogen bonds in a reverse wobble conformation with the other pyrimidine-like component, and

a base-specific nucleobase complementary to a single nucleobase variation at its second polymorphic site, wherein the base-specific nucleobasc is characteristic of a single allele of the polynucleotide template;

and wherein the hydrogen bonding pattern of the degenerate nucleobase of the oligomer is pyAAD or puDDA.

2. The polynucleotide duplex according to claim 1, wherein the oligomer comprises a plurality of degenerate nucleobases, each of which is complementary to multiple natural nucleobase variations at a corresponding polymorphic site of the corresponding polynucleotide template.

3. The polynucleotide duplex according to claim 1, wherein a melting temperature of the degenerate nucleobase oligomer and the polynucleotide template is substantially equivalent to a melting temperature of a natural nucleobase oligomer and the polynucleotide template.

4. The polynucleotide duplex according to claim 1, wherein the degenerate nucleobase of the oligomer is flanked by natural nucleobases.

5. The polynucleotide duplex according to claim 1, wherein the hydrogen bonding pattern of the degenerate nucleobase with a corresponding nucleobase of the polynucleotide template is puDDA:

pyDAA, puDDA;

pyADA, puDA;

pyAAD, or puADD;

pyAAD.

6. The polynucleotide duplex according to claim 1, wherein the degenerate nucleobase having a hydrogen bonding pattern of pyAAD is 5-methylisocytidine, isocytidine, or 2′

-deoxy analogs thereof.

7. The polynucleotide duplex according to claim 1, wherein the degenerate nucleobase having a hydrogen bonding pattern of puDDA is isoguanosine, 7-deazaisoguanosine, 7-deaza-8-azaisoguanosine, N6-(6-aminohexyl)isoguanosine, N6-(2-(1H,imidazol-4-y1)-ethyl)isoguanosine, N6-(N-(Dabcy 1)-6-aminohexyl)isoguanosine, or 2′

-deoxy analogs thereof.

8. The polynucleotide duplex according to claim 1, wherein the base-specific nucleobase hybridizes selectively to each allele of a target polymorphic locus.

9. The polynucleotide duplex according to claim 1, wherein the base-specific nucleobase is non-complementary to a single allele of a polymorphic locus.

10. The polynucleotide duplex according to claim 1, wherein the base-specific nucleobase is complementary to a single allele of a polymorphic locus.