Epoch Pharmaceuticals, Inc.

Epoch Biosciences, Inc.

Cepheid Corporation

Other

Elitech Holding B.v.

Igor V Kutyavin

Rich B Meyer, Jr

Irina Aleksandrovna Afonina

Eugeny Alexander Lukhtanov

Alexander A Gall

Robert O Dempcy

Howard B Gamper

Sergey G Lokhov

Nicolaas M J Vermeulen

Joel Hedgpeth

Evgeniy S Belousov

Michael J Singer

Merl F Hoekstra

David Milesi

Yevgeniy S Belousov

Michael W Reed

Charles R Petrie

John C Tabone

Rich B Meyer

Gerald D Hurst

Karen M Singer, Legal Representative

J Ansel Wald

Jinsuk Woo

Nicolaas MJ Vermeulen

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising modifying the aptamer by uracil-DNA glycosylase enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. In some aspects, the oligonucleotide aptamers are circular and comprise one or more deoxyuridine nucleotides providing for aptamer-specific recognition and modification of the circular aptamer by the uracil-DNA glycosylase enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing uracil-DNA glycosylase enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one uracil-DNA glycosylase enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific modification of the aptamer by the uracil-DNA glycosylase enzymatic activity.

Inhibition of DNA polymerases by uracil-DNA glycosylase-cleavable oligonucleotide ligands

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. The oligonucleotide aptamers of the present invention are circular and comprise one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the circular aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.

Inhibition of nucleic acid polymerases by endonuclease V-cleavable circular oligonucleotide ligands

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. In some aspects, the oligonucleotide aptamer comprises one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.

Inhibition of nucleic acid polymerases by endonuclease V-cleavable oligonucleotide ligands

Inhibition of nucleic acid polymerases by endonuclease v-cleavable oligonucleotide ligands

Provided are nucleic acid detection methods wherein targeted primer extension or products or amplification products incorporate a probe-anchoring modification introduced using a primer incorporating a probe-anchoring primer modification, wherein oligonucleotide detection probes incorporate a probe-anchoring probe modification, the primers and probes designed to place the binding site of the oligonucleotide probe proximate to the probe-anchoring primer modification in the detected target sequence. The probe-anchoring modifications of the probe and the primer-extension product form a stabilized complex comprising a detectible duplex of the probe with the detected target sequence. In certain aspects, the probe-anchoring modified primer also incorporates a probe-directing sequence. The methods allow use of exceptionally short oligonucleotide probes (e.g., 10-mer and shorter) enabling establishment of a complete probe inventory or universal library containing a probe complementary to any target nucleic acid sequence. Real-time and post-amplification detection methods are provided, along with detection kits.

Methods and compositions for detection of nucleic acids based on stabilized oligonucleotide probe complexes

Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, the methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences. During target sequence amplification in the presence of DNA polymerase activity, a FEN1 activity, one or more amplification primers, deoxynucleoside 5'-triphosphates and other reagents suitable to support amplification of both target and homologous nucleic acid sequences, the cleavage directing oligonucleotide hybridizes to the homologous sequence and its amplification products resulting in a FEN1-mediated cleavage of these sequences providing for increased amounts of the target nucleic acid sequence relative to that of the homologous nucleic acid sequences.

Methods and compositions for enrichment of nucleic acids in mixtures of highly homologous sequences

Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5'-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing the oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3&#x2032;- or 5&#x2032;-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid. In certain aspects both primers incorporate a 5&#x2032;-specialty sequence and detecting comprises cleaving 3&#x2032;- or 5&#x2032;-strands of a three-strand cleavage structure using duplex-specific nuclease to provide a cleavage product.

Use of products of PCR amplification carrying elements of secondary structure to improve PCR-based nucleic acid detection

The present invention provides compositions, methods and kits for use in the detection of small RNA sequences, which allow for rapid and robust amplification and detection. The methods provide improved sensitivity and efficiency in the amplification-based detection of small RNA sequences by incorporating one or more base-modified duplex-stabilizing dNTPs during reverse transcription and/or amplification.

Methods, compositions and kits for the improved detection of small RNA molecules

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPIgroup 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.

Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

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Woodinville, WA, United States of America

Igor V Kutyavin