Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI 3 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.
1. A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, the method comprising:
(a) contacting the mixture of polynucleotides with at least one Minor Groove Binder (MGB)-oligonucleotide conjugate,
wherein the MGB-oligonucleotide conjugate comprises a 5′-end, a 3′-end, a Minor Groove Binder (MGB), one or more detectable fluorescent labels, and a quenching agent which quenches the fluorescence emission of one or more of the fluorescent labels; and
(b) measuring hybrid formation, whereby hybrid formation is indicative of the presence of said target sequence.
2. The method of claim 1 , further comprising the step of releasing one or more labels from the MGB-oligonucleotide conjugate subsequent to hybrid formation.
3. The method of claim 1 , wherein the detectable labels are fluorescein, cyanine, rhodamine, or combinations thereof.
4. The method of claim 1 , wherein at least one of the detectable labels is a fluorescein.
5. The method of claim 1 , wherein the quenching agent is tetramethylrhodamine.
6. The method of claim 1 , wherein the MGB-oligonucleotide conjugate comprises at least two fluorescent labels.
7. The method of claim 6 , wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.
8. The method of claim 1 , wherein the MGB is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 10 3 M −1 .
9. The method of claim 1 , wherein the MGB is located at the 5′ end of the MGB-oligonucleotide conjugate.
10. The method of claim 1 , wherein the MGB is located at the 3′ end of the MGB-oligonucleotide conjugate.
11. The method of claim 1 , wherein at least one of the detectable fluorescent labels is located at the 5′ end of the MGB-oligonucleotide conjugate.
12. The method of claim 1 , wherein at least one of the detectable fluorescent labels is located at the 3′ end of the MGB-oligonucleotide conjugate.
13. The method of claim 1 , wherein the MGB is selected from the group consisting of a dimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 2 ), a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ), tetramer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 4 ) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC 5 ).
14. The method of claim 1 , further comprising the step of altering the spatial relationship between at least one of the detectable fluorescent labels and the quenching agent.
15. The method of claim 1 , wherein the MGB-oligonucleotide conjugate is a primer in an amplification reaction.
16. The method of claim 15 , wherein the amplification reaction is a polymerase chain reaction.