US 2003/0198973 A1 · Chimeric fusion molecule for analyte detection and quantitation

Abstract

Detector fusion molecules are produced by attaching a protein sub-unit to a linker, and attaching the linker to a nucleic acid molecule. The detector fusion molecules have utility in detecting and quantifying a specific target analyte from a sample. The protein sub-unit of the detector fusion molecule is selected to specifically bind the specific target analyte. The nucleic acid molecule of the detector fusion molecule is used as a tag, thus allowing for the detection and quantification of the target analyte. The sample is contacted with detector fusion molecules, thereby allowing detector fusion molecules to specifically bind any specific target analytes in the sample. The nucleic acid molecule of the detector fusion molecule is amplified using known processes, thereby producing an amplification product. The amplification product is detected and quantified, thus determining an amount of the target analyte in the sample.

Claims117

Dependent claims

A method for producing a fusion molecule capable of use as a detector molecule for binding a predetermined target analyte, said method comprising the steps of:

(a) attaching a reactive moiety to a first end of a protein sub-unit, thereby creating a reactive intermediate with said reactive moiety at a first end thereof;
(b) bonding a coupling reagent to a first end of a nucleic acid molecule, thereby forming a modified nucleic acid molecule, said coupling reagent of said modified nucleic acid molecule being capable of displacing said reactive moiety of said reactive intermediate;
and
(c) catalyzing a reaction between said reactive moiety of said reactive intermediate and said coupling reagent of said modified nucleic acid molecule, wherein in said reaction:
(i) said reactive moiety is displaced from said first end of said reactive intermediate;
and
(ii) a covalent bond is formed between said first end of said reactive intermediate and said first end of said modified nucleic acid molecule.

4 more elements

A method for producing a fusion molecule capable of use as a detector molecule for binding a predetermined target analyte, said method comprising the steps of:

(a) attaching a reactive moiety to a first end of a protein sub-unit, thereby creating a reactive intermediate with said reactive moiety at a first end thereof;
(b) attaching a cysteine-like moiety to a nucleotide, thereby forming a modified nucleotide;
(c) linking a first end of a nucleic acid molecule to said modified nucleotide, thereby forming a modified nucleic acid molecule, said cysteine-like moiety of said modified nucleic acid molecule being capable of displacing said reactive moiety of said reactive intermediate;
and
(d) catalyzing a reaction between said reactive moiety of said reactive intermediate and said cysteine-like moiety of said modified nucleic acid molecule, wherein in said reaction:
(i) said reactive moiety is displaced from said first end of said reactive intermediate;
and
(ii) a covalent bond is formed between said first end of said reactive intermediate and said first end of said modified nucleic acid molecule.

5 more elements

A method for producing a fusion molecule capable of use as a detector molecule for binding a predetermined target analyte, said method comprising the steps of:

(a) attaching reactive moieties to first ends of protein sub-units of a quantity of protein sub-units, thereby creating a quantity of reactive intermediates with said reactive moieties at first ends thereof;
(b) bonding first coupling reagents to first nucleotides, thereby forming first modified nucleotides;
(c) connecting second coupling reagents to second nucleotides, thereby forming second modified nucleotides;
(d) linking first ends of a quantity of nucleic acid molecules to said first modified nucleotides, and linking second ends of said quantity of said nucleic acid molecules to said second modified nucleotides, thereby forming modified nucleic acid molecules with first and second ends;
(e) severing said modified nucleic acid molecules between said first and said second ends of said nucleic acid thereof, thereby forming:
(i) from said first end from said modified nucleic acid first modified nucleic acid fragments containing said first modified nucleotide;
and
(ii) from said second end from said modified nucleic acid second modified nucleic acid fragments containing said second modified nucleotide;
and
(f) catalyzing a first reaction between said first coupling reagent of said first modified nucleic acid fragments and reactive moieties of said reactive intermediates of said quantity thereof, wherein in said reaction:
(i) said reactive moieties are displaced from said first ends of said reactive intermediates;
and
(ii) first covalent bonds are formed between said reactive intermediate and said first modified nucleotide of said first modified nucleic acid fragment;
(g) catalyzing a second reaction between said second coupling reagent of said second modified nucleic acid fragments and reactive moieties of said reactive intermediates of said quantity thereof, wherein in said reaction:
(i) said reactive moieties are displaced from said reactive intermediates;
and
(ii) second covalent bonds are formed between said reactive intermediates and said second modified nucleotides of said second modified nucleic acid fragments.

14 more elements

The product of a process comprising:

(a) attaching a reactive moiety to a first end of a protein sub-unit, thereby creating a reactive intermediate with said reactive moiety attached at a first end thereof;
(b) bonding first coupling reagents to first nucleotides, thereby forming first modified nucleotides;
(c) connecting second coupling reagents to second nucleotides, thereby forming second modified nucleotides;
(d) linking first ends of a quantity of nucleic acid molecules to said first modified nucleotides, and linking second ends of said quantity of said nucleic acid molecules to said second modified nucleotides, thereby forming modified nucleic acid molecules with first and second ends;
(e) severing said modified nucleic acid molecules between said first and said second ends of said nucleic acid molecules thereof, thereby forming:
(i) from said first end from said modified nucleic acid molecule first modified nucleic acid fragments containing said first modified nucleotide;
and
(ii) from said second end from said modified nucleic acid molecule second modified nucleic acid fragments containing said second modified nucleotide;
and
(f) catalyzing a first reaction between said first coupling reagent of said first modified nucleic acid fragments and reactive moieties of said reactive intermediates of said quantity thereof, wherein in said reaction:
(i) said reactive moiety is displaced from said first end of said reactive intermediate;
and
(ii) a first covalent bond is formed between said reactive intermediate and said first modified nucleotide of said first modified nucleic acid fragment;
(g) catalyzing a second reaction between said second coupling reagent of said second modified nucleic acid fragments and reactive moieties of said reactive intermediates of said quantity thereof, wherein in said reaction:
(i) said reactive moieties are displaced from said reactive intermediates;
and
(ii) second covalent bonds are formed between said reactive intermediates and said second modified nucleotides of said second modified nucleic acid fragments.

14 more elements

A method for recognizing a target analyte in a sample, said method comprising the steps of:

(a) manufacturing a quantity of detector fusion molecules, said step of manufacturing comprising the steps of:
(i) attaching a quantity of reactive moieties to a first end of a quantity of protein sub-units capable of binding to the target analyte, thereby creating a quantity of reactive intermediates with said reactive moieties at first ends thereof;
(ii) bonding a quantity of coupling reagents to first ends of a quantity of nucleic acid molecules, thereby forming a quantity of modified nucleic acid molecules, said coupling reagent of said modified nucleic acid molecules being capable of displacing said reactive moiety of said reactive intermediates;
and
(iii) catalyzing a reaction between said reactive moiety of said reactive intermediates and said coupling reagent of said modified nucleic acid molecules, wherein in said reaction:
(A) said reactive moiety is displaced from said first ends of said reactive intermediates;
and
(B) a covalent bond is formed between said first end of said reactive intermediates and said first end of said modified nucleic acid molecules;
(b) contacting the sample with said quantity of said detector fusion molecules, whereby said detector fusion molecules from said quantity thereof bind to the target analyte in the sample;
(c) amplifying said nucleic acid molecules of said detector fusion molecules bound to the target analyte, thereby producing an amplification product;
and
(d) identifying said amplification product.

9 more elements

A method for quantifying a target analyte in a sample, said method comprising the steps of:

(a) manufacturing a quantity of detector fusion molecules, said step of manufacturing comprising:
(i) attaching a quantity of reactive moieties to a first end of a quantity of protein sub-units capable of binding to the target analyte, thereby creating a quantity of a reactive intermediates with said reactive moieties at first ends thereof;
(ii) bonding a quantity of coupling reagents to a first ends of a quantity of nucleic acid molecules, thereby forming a quantity of modified nucleic acid molecules, said coupling reagent of said modified nucleic acid molecule being capable of displacing said reactive moiety of said reactive intermediates;
and
(iii) catalyzing a reaction between said reactive moiety of said reactive intermediates and said coupling reagent of said modified nucleic acid molecules, wherein of said reaction:
(A) said reactive moiety is displaced from said first ends of said reactive intermediates;
and
(B) a covalent bond is formed between said first end of said reactive intermediates and said first end of said modified nucleic acid molecules;
(b) contacting the sample with said quantity of said detector fusion molecules, whereby said detector fusion molecules from said quantity thereof bind to the target analyte in the sample;
(c) amplifying said nucleic acid molecules of said quantity of said detector fusion molecules bound to the target analyte, thereby producing an amplification product;
and
(d) determining an amount of said amplification product.

9 more elements

A method for creating a nanostructure on a target analyte using a detector fusion molecule, said method comprising the steps of:

(a) manufacturing a quantity of detector fusion molecules, said step of manufacturing comprising:
(i) attaching a quantity of reactive moieties to a first end of a quantity of protein sub-units capable of binding to the target analyte, thereby creating a quantity of a reactive intermediates with said reactive moieties at first ends thereof;
(ii) bonding a quantity of coupling reagents to a first ends of a quantity of nucleic acid molecules, thereby forming a quantity of modified nucleic acid molecules, said coupling reagent of said modified nucleic acid molecule being capable of displacing said reactive moiety of said reactive intermediates;
and
(iii) catalyzing a reaction between said reactive moiety of said reactive intermediates and said coupling reagent of said modified nucleic acid molecules, wherein of said reaction:
(A) said reactive moiety is displaced from said first ends of said reactive intermediates;
and
(B) a covalent bond is formed between said first end of said reactive intermediates and said first end of said modified nucleic acid molecules;
(b) attaching a target analyte to a substrate, thereby forming a multimeric complex;
(c) binding said detector fusion molecule to said multimeric complex, thereby forming a fusion molecule-analyte complex;
and
(d) linking a higher order structure to said fusion molecule-analyte complex.

9 more elements

Dependencies are inferred from the claim text.

Chimeric fusion molecule for analyte detection and quantitation

Life of this patent

Chimeric fusion molecule for analyte detection and quantitation

Life of this patent

Abstract

Claims117