Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-04
2002-12-03
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S069100, C435S068100, C536S023100, C536S023400, C536S024300, C536S024330, C436S536000, C436S518000
Reexamination Certificate
active
06489116
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, the invention relates to diagnostic methods involving multiplex analysis.
A variety of methods exist to detect multiple species in a biological sample. These include ELISA based immunoabsorbent assays, protein biochips, and the like. Each of these methods suffers from limitations in detection sensitivity or selectivity, due, for example, to kinetics of binding or sensitivity of detection reagents. In addition, these techniques are also limited in terms of the number of molecules that can be rapidly detected.
SUMMARY OF THE INVENTION
The present invention involves a novel multiplex diagnostic approach for the ultra-sensitive detection of molecules in biological samples.
In general, in a first aspect, the invention features a method for detecting multiple compounds in a sample, the method involving: (a) contacting the sample with a mixture of binding reagents, the binding reagents being nucleic acid-protein fusions, each having (i) a protein portion which is known to specifically bind to one of the compounds and (ii) a nucleic acid portion which encodes the protein portion and which includes a unique identification tag; (b) allowing the protein portions of the binding reagents and the compounds to form complexes; (c) capturing the binding reagent-compound complexes; (d) amplifying the nucleic acid portions of the complexed binding reagents; and (e) detecting the unique identification tag of each of the amplified nucleic acids, thereby detecting the corresponding compounds in the sample.
In preferred embodiments, the sample is a biological sample; the nucleic acid-protein fusion is an RNA-protein fusion; the nucleic acid-protein fusion is covalently bound; the nucleic acid-protein fusion is covalently bound through a peptide acceptor; the peptide acceptor is puromycin; the binding reagents do not bind the compounds through compound-specific antibody domains; each of the binding reagents includes a scaffold domain; each of the binding reagents includes a fibronectin scaffold domain; the fibronectin scaffold domain is the 10
th
domain of fibronectin type III; each of the binding reagents includes an antibody scaffold domain; the binding reagents bind the compounds with equilibrium constants of less than about 500 nM; the unique identification tags are detected using a solid support to which are immobilized nucleic acids specific for the unique identification tags and the detection is accomplished by hybridization of the unique identification tags to the immobilized nucleic acids; the amplifying step (d) is carried out using quantitative PCR; the compounds are proteins; the mixture of binding reagents includes at least 5 different nucleic acid-protein fusions, each specifically binding to a different compound; the mixture of binding reagents includes at least 100 different nucleic acid-protein fusions, each specifically binding to a different compound; the mixture of binding reagents includes at least 40,000 different nucleic acid-protein fusions, each specifically binding to a different compound; and/or the mixture of binding reagents includes at least 500,000 different nucleic acid-protein fusions, each specifically binding to a different compound.
In a second aspect, the invention features a method for detecting a compound in a sample, the method involving: (a) contacting the sample with a binding reagent, the binding reagent being a nucleic acid-protein fusion having (i) a protein portion which is known to specifically bind to the compound and (ii) a nucleic acid portion which encodes the protein portion and which includes a unique identification tag; (b) allowing the protein portion of the binding reagent and the compound to form a complex; (c) capturing the binding reagent- compound complex; (d) amplifying the nucleic acid portion of the complexed binding reagent; and (e) detecting the unique identification tag of the amplified nucleic acid, thereby detecting the corresponding compound in the sample.
In a related aspect, the invention features a kit for carrying out compound detection, the kit including: (a) a nucleic acid-protein fusion, wherein the protein portion of the fusion specifically binds the compound and the nucleic acid portion of the fusion encodes the protein portion and includes a unique identification tag; (b) a PCR primer pair, wherein the first of the primers hybridizes to the nucleic acid portion of the fusion 5′ to the unique identification tag and the second of the primers hybridizes to the nucleic acid portion of the fusion 3′ to the unique identification tag and hybridization of the primers to the nucleic acid fusion permits amplification of the unique identification tag; and (c) a solid support including a nucleic acid which can hybridize to the unique identification tag.
In preferred embodiments, the kit further includes Taq polymerase; the nucleic acid-protein fusion is an RNA-protein fusion; the nucleic acid-protein fusion is covalently bound; the nucleic acid-protein fusion is covalently bound through a peptide acceptor; the peptide acceptor is puromycin; the nucleic acid- protein fusion does not bind the compound through a compound-specific antibody domain; the nucleic acid-protein fusion includes a scaffold domain; the nucleic acid-protein fusion includes a fibronectin scaffold domain; the fibronectin scaffold domain is the 10
th
domain of fibronectin type III; the nucleic acid-protein fusion includes an antibody scaffold domain; the nucleic acid-protein fusion binds the compound with an equilibrium constant of less than about 500 nM; the solid support is a chip; the solid support includes an ordered array of single-stranded nucleic acids on its surface, each of the single-stranded nucleic acids being capable of hybridizing to a different unique identification tag; the compound is a protein; the kit includes at least 5 different nucleic acid-protein fusions, each specifically binding to a different compound; the kit includes at least 100 different nucleic acid-protein fusions, each specifically binding to a different compound; the kit includes at least 40,000 different nucleic acid-protein fusions, each specifically binding to a different compound; and/or the kit includes at least 500,000 different nucleic acid-protein fusions, each specifically binding to a different compound.
According to this approach, one begins with a set of uniquely defined high affinity binding reagents (typically protein binding reagents). Each of these reagents binds to a different target in a sample, facilitating the detection of several targets simultaneously. The targets of the binding reagents are frequently proteins, but they may be any moiety capable of specific binding, including, for example, nucleic acids or sugar moieties. Such binding reagents may represent naturally-occurring or partially or completely synthetic amino acid sequences. Examples of naturally-occurring binding reagents include, without limitation, members of the following binding pairs: antigen/antibody pairs, protein/inhibitor pairs, receptor/ligand pairs (for example cell surface receptor/ligand pairs, such as hormone receptor/peptide hormone pairs), enzyme/substrate pairs (for example, kinase/substrate pairs), lectin/carbohydrate pairs, oligomeric or heterooligomeric protein aggregates, DNA binding protein/DNA binding site pairs, RNA/protein pairs, and nucleic acid duplexes, heteroduplexes, or ligated strands, as well as any molecule which is capable of forming one or more covalent or non-covalent bonds (for example, disulfide bonds) with any portion of a nucleic acid-protein fusion. In addition to naturally-occurring binding partner members, binding reagents may be derived by any technique, for example, by directed evolution approaches using a desired protein as the binding target.
Whether naturally-occurring or synthetic, when mixtures of binding reagents are utilized in a single diagnostic reaction mixture, they are preferably similar in composition and amino acid length. In a particularly preferred approach, one starts with a common ami
Clark & Elbing LLP
Guzo David
Leffers, Jr. Gerald G.
Phylos, Inc.
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