Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

• US 6,884,584 B2
• Filed: 04/22/2002
• Issued: 04/26/2005
• Est. Priority Date: 04/03/1995
• Status: Expired due to Fees

• Current Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Meyer, Rich B. Jr., Lukhtanov, Eugeny A., Kutyavin, Igor V., Hedgpeth, Joel, Belousov, Evgeniy S., Afonina, Irina A.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US10/127,936
• Publication Number:
  US 20040058322A1
• Time in Patent Office: 1,100 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/26.6
• US Class Current: 435/6.1
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI₃ group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

Claims

1. A method for identifying one or more nucleotide polymorphisms in a polynucleotide sample, the method comprising:

providing an array of support-bound oligonucleotide probes of different sequences;

incubating a polynucleotide sample with said array under hybridization conditions;

incubating a plurality of different minor groove binder-oligonucleotide conjugate probes with said polynucleotide sample with said array under hybridization conditions to form one or more minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids,

wherein said minor groove binder is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds in a non-intercalating manner into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 10³M⁻

; and

detecting the presence of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids on said array.

2. The method of claim 1, wherein the nucleotide polymorphisms are one or more single-nucleotide polymorphisms.

3. The method of claim 1, wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 12.5% to 100%.

4. The method of claim 3, wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 18°

C.

5. The method of claim 1, wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 25% to 87.5%.

6. The method of claim 5, wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 8°

C.

7. The method of claim 1, wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 37.5% to 75%.

8. The method of claim 7, wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 4°

C.

9. The method of claim 1, wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 37.5% to 87.5%.

10. The method of claim 9, wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 4°

C.

11. The method of claim 1, wherein said minor groove binder is a member selected from the group consisting of:

netropsin;

distamycin;

lexitropsin;

mithramycin;

chromomycin A₃;

olivomycin;

anthramycin;

sibiromycin;

pentamidine;

stilbamidine;

berenil;

CC-1065;

Hoechst 33258;

4′

-6-diamidino-2-phenylindole (DAPI);

a dimer, a trimer, a tetramer and a pentamer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI);

N-methylpyrrole-4-carbox-2-amide (MPC), and a dimer, a trimer, a tetramer and a pentamer of MPC.

12. The method of claim 1, wherein said minor groove binder-oligonucleotide conjugate probe(s) contain a detectable label.

13. The method of hybridization according to claim 1, wherein the melting temperature of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is relatively independent of base composition.

14. The method of hybridization according to claim 1 wherein the melting temperature of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is dependent primarily on the length of the hybridized nucleic acid.