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US 2003/0113716 A1Published application
Nucleic acid multiplex formation
AssigneeIngeneus Corporation
InventorsGlen H. Erikson, Jasmine I. Daksis, Ivana Kandic, Pierre Picard
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Abstract
Heteropolymeric triplexes and quadruplexes and methods for making them; the use of accelerator agents such as cations to create them; the use of fluorescent intercalators and fluorescent probe-bound non-intercalators to detect them.
Claims55
A method of creating a nucleic acid multiplex, said method comprising the steps of:
- 1) creating a mixture comprising water, a Watson-Crick duplex, a sufficient number of single-stranded mixed base sequence molecules to form a multiplex that includes the Watson-Crick duplex, and an accelerator agent that increases a rate or amount of multiplex formation, said multiplex being a triplex or quadruplex, wherein said single-stranded molecule or molecules are selected so that, if in a multiplex, they would each be related to all other strands of the multiplex by adherence to base pairing rules, said rules being either Watson-Crick base-pairing rules or homologous binding base-pairing rules;
- and
- 2) incubating said mixture to allow the multiplex to form, each strand of said multiplex related to all other strands of the multiplex by adherence to base-pairing rules;
- provided that, within the multiplex, the Watson-Crick duplex added in step (1) is heteropolymeric with a G-C content between 10% and 90%.
1 more element
A triplex comprising a single-stranded probe bound to a double-stranded nucleic acid target, wherein said probe comprises a heteropolymeric nucleic acid or a heteropolymeric nucleic acid analog, and all base triplets of said triplex are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C.
A quadruplex comprising:
- a first strand containing a first sequence of nucleobases;
- a second strand containing a second sequence of nucleobases, wherein said second strand is associated with said first strand by Watson-Crick bonding;
- a third strand containing a third sequence of nucleobases;
- and
- a fourth strand containing a fourth sequence of nucleobases, wherein said fourth strand is associated with said third strand by Watson-Crick bonding.
2 more elements
A method of detecting a triplex, said method comprising:
- providing a target double-stranded nucleic acid or nucleic acid analogue comprising a target sequence, wherein said target sequence contains at least one purine base and at least one pyrimidine base;
- providing a probe comprising a nucleic acid sequence or a nucleic acid analog sequence;
- providing an accelerator agent;
- adding said probe, said target sequence and said accelerator agent to a medium to provide a test sample containing a triplex complex comprising said probe bound to said target sequence, wherein all base triplets of said complex are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C;
- irradiating said test sample with exciting radiation to cause the test sample to emit fluorescent radiation;
- detecting an intensity of said fluorescent radiation, wherein said intensity is correlated with a binding affinity between said probe and said target sequence;
- and
- determining from said intensity an extent of matching between said probe and said target sequence;
- wherein said method is a homogeneous assay conducted without providing a signal quenching agent on said target sequence or on said probe.
6 more elements
A method of detecting a triplex, said method comprising:
- providing a target nucleic acid or nucleic acid analogue having a target sequence, wherein said target sequence contains at least one purine base and at least one pyrimidine base;
- providing a double-stranded probe comprising a nucleic acid sequence or a nucleic acid analog sequence;
- providing a hybridization accelerator agent;
- adding said probe, said target and said hybridization accelerator agent to a medium to provide a test sample containing a Watson-Crick triplex comprising said probe bound to said target sequence;
- irradiating said test sample with exciting radiation to cause test sample to emit fluorescent radiation;
- detecting an intensity of said fluorescent radiation, wherein said intensity is correlated with a binding affinity between said probe and said target sequence;
- and
- determining from said intensity an extent of matching between said probe and said target sequence
- wherein said method is a homogeneous assay conducted without providing a signal quenching agent on said target sequence or on said probe.
6 more elements
A method of detecting a quadruplex, said method comprising:
- providing a target nucleic acid or nucleic acid analogue having a target sequence, wherein said target sequence contains at least one purine base and at least one pyrimidine base;
- providing a double-stranded probe comprising a nucleic acid sequence or a nucleic acid analog sequence;
- providing a hybridization accelerator agent;
- adding said probe, said target and said hybridization accelerator agent to a medium to provide a test sample containing a Watson-Crick quadruplex comprising said probe bound to said target sequence;
- irradiating said test sample with exciting radiation to cause test sample to emit fluorescent radiation;
- detecting an intensity of said fluorescent radiation, wherein said intensity is correlated with a binding affinity between said probe and said target sequence;
- and
- determining from said intensity an extent of matching between said probe and said target sequence
- wherein said method is a homogeneous assay conducted without providing a signal quenching agent on said target sequence or on said probe.
6 more elements
Dependencies are inferred from the claim text.