Conjugates of nucleotides and method for the application thereof

A method for enzymatic synthesis of nucleic acid chains, wherein nucleotides are enzymatically incorporated into the complementary strand of at least one target sequence or equivalents thereof, wherein at least one of the nucleotides used has the following structure:

• (Nuc-linker)n-marker
• wherein:
• Nuc is a nucleotide (nuc-component)
• Linker is a linker component wherein the linker links the nuc-component to the macromolecular marker component
• Marker is a marker component which comprises at least one target domain and at least one anchor domain
• n is a positive integer from 1 to 1000

A method for enzymatic synthesis of nucleic acid chains, wherein nucleotides are enzymatically incorporated into the complementary strand of at least one target sequence or equivalents thereof, wherein at least one of the used nucleotides has the following structure:

• (Nuc-linker)n-marker
• wherein:
• Nuc is a nucleotide (nuc-component)
• Linker is a linker component wherein the linker links the nuc-component to the macromolecular marker component
• Marker is a marker component which comprises at least one target domain and at least one signal domain
• n is a positive integer from 1 to 1000

A method for enzymatic synthesis of nucleic acid chains, wherein nucleotides are enzymatically incorporated into the complementary strand of at least one target sequence or equivalents thereof, wherein at least one of the used nucleotides has the following structure:

• (Nuc-linker)n-marker
• wherein:
• Nuc is a nucleotide (nuc-component)
• Linker is a linker component wherein the linker links the nuc-component to the macromolecular marker component
• Marker is a marker component which comprises at least one target domain and at least one anchor domain and at least one signal domain
• n is a positive integer from 1 to 1000

A method for enzymatic synthesis of nucleic acid chains, wherein nucleotides are enzymatically incorporated into the complementary strand of at least one target sequence or equivalents thereof, wherein at least one of the used nucleotides has the following structure:

• (Nuc-linker)n-marker
• wherein:
• Nuc is a nucleotide (nuc-component)
• Linker is a linker component wherein the linker links the nuc-component to the macromolecular marker component
• Marker is a marker component which comprises at least one anchor domain
• n is a positive integer from 1 to 1000

A method for the enzymatic synthesis of nucleic acid chains comprising the following steps:

• Performing an enzymatic synthesis of at least one nucleic acid chain (the target sequence) with at least one polymerase, with at least one primer that is capable of binding to the specific target sequence, and with at least one conjugate type (one type of nuc-macromolecule),
• said conjugate (nuc macromolecule) comprises at least one nucleoside triphosphate or its analog, which can be recognized by a polymerase as a substrate, and at least one oligonucleotide, wherein the oligonucleotide is capable of specifically hybridizing to the target sequence,
• said polymerase incorporates the said nucleoside triphosphate or its analog during the enzymatic synthesis into the growing strand complementary to the target sequence,
• Detection of the incorporation event of the conjugate (nuc macromolecule)

A method for the enzymatic synthesis of nucleic acid chains comprising the following steps:

• Performing an enzymatic synthesis of at least one nucleic acid chain (the target sequence) with at least one polymerase, with at least one primer that is capable of binding to the specific target sequence, with at least one conjugate type (one type of nuc-macromolecule),
• said conjugate type (nuc macromolecule) comprises at least one nucleoside triphosphate or its analog, which can be recognized by a polymerase as a substrate, and at least one oligonucleotide, wherein the oligonucleotide is capable of specifically hybridizing to the target sequence downstream from the 3′ position of the primer,
• said polymerase incorporates said nucleoside triphosphate or its analog into the growing strand complementary to the target sequence during the enzymatic synthesis,
• Detection of the incorporation event of the conjugate (nuc macromolecule)

A method for the enzymatic synthesis of nucleic acid chains comprising the following steps:

• Performing an enzymatic synthesis of at least one nucleic acid chain (the target sequence) with at least one polymerase, with at least one primer that is capable of binding to the specific target sequence, with at least one conjugate type (one type of nuc-macromolecule) and at least one hybridizing probe
• said conjugate type (nuc macromolecule) comprises at least one nucleoside triphosphate or its analog, which can be recognized by a polymerase as a substrate, and at least one oligonucleotide, wherein the oligonucleotide is capable of specifically hybridizing to the target sequence downstream from the 3′ position of the primer,
• said hybridizing probe comprises at least one oligonucleotide, wherein the oligonucleotide is capable of specifically hybridizing to the target sequence downstream from the 3′ position of said oligonucleotide of the conjugates (nuc macromolecule)
• said polymerase incorporates said nucleoside triphosphate or its analog into the growing strand complementary to the target sequence during the enzymatic synthesis,
• Detection of the incorporation event of the conjugate (nuc macromolecule)

A method for the enzymatic synthesis of nucleic acid chains comprising the following steps:

• Incubating of at least one nucleic acid chain (the target sequence) with at least one polymerase, with at least one primer that is capable of binding to the specific target sequence, with at least one conjugate type (one type of nuc-macromolecule)
• said conjugate type (nuc macromolecule) comprises at least one nucleoside triphosphate or its analog, which can be recognized by a polymerase as a substrate, and at least one oligonucleotide, wherein the oligonucleotide is capable of specifically hybridizing to the target sequence
• Incubation under conditions which allow for specific hybridization of primers and/or conjugate (and/or optionally a hybridization probe) to the nucleic acid chain (the target sequence)
• Incubation under conditions which allow an enzymatic coupling of the conjugate by the polymerase. Said polymerase incorporates the nucleoside triphosphate or its analog into the growing strand complementary to the target sequence during the enzymatic synthesis,
• Detection of the incorporation event of the conjugate (nuc macromolecule)

A method for amplification (PCR) comprising the following steps:

• Providing a PCR with at least one nucleic acid chain (the target sequence) from a sample, with at least one polymerase, with at least two PCR primers that are capable of binding to the specific target sequence, with at least one conjugate type (one type of nuc-macromolecule)
• said conjugate type (nuc macromolecule) comprises at least one nucleoside triphosphate or its analog, which can be recognized by said polymerase as a substrate, and at least one oligonucleotide, wherein the oligonucleotide is capable of specifically hybridizing to the target sequence between the two PCR primers
• Providing of oligonucleotide primers, wherein the one primer has a complementary sequence to one strand of the target sequence and can start an extension reaction and the other primer has a complementary sequence to the other strand of the target sequence and can start an extension reaction, whereby both primers are selected so that their binding sites within the target sequence are located on either side of the binding site of said conjugate (nuc-macromolecule)
• Amplification of the target sequence, if present in the sample, using a polymerase and conditions of a PCR:
• (a) hybridization of the primers and of the conjugate to the target sequence, (b) extension of the primers and incorporation of conjugates (c) where appropriate, separation of the synthesized double strands of the target sequence, (d) where appropriate, repeating steps (a) to (c) several times
• Detection of the incorporation event of the conjugate (nuc macromolecule)

Conjugate (called a nuc-macromolecule) which comprises at least the following components:

• At least one nucleotide
• At least one oligonucleotide
• At least one linker
• wherein the nucleotide is a nucleoside triphosphate or its analog which is able to be incorporated into a primer by a polymerase,
• the oligonucleotide is capable of binding to a target sequence,
• the linker is placed between the nucleoside triphosphate and the oligonucleotide

A kit with components for the manufacturing of novel said macromolecules. This kit comprises at least one of the following components:

• At least one reactive nuc-component or nuc-linker component which can be coupled to the oligonucleotides.
• For example, components of such a kit are nuc-components or nuc-linker components with an amino-reactive group (for example nuc-components with a NHS ester or an isothiocyanate) or a thiol-reactive group (e.g. nuc-components with a maleimide group).
• At least one oligonucleotide having a sequence capable of binding specifically to the target sequence
• Buffer solutions for coupling nuc-components to the oligonucleotides