Patent Application
20060141505
1. The Field of the Invention
The invention disclosed herein relates generally to the assay of samples of molecules involved in a biological activity. More specifically, the present invention relates to detector molecules of the type used to detect and quantify target analytes in such samples. The present invention has particular applicability to the detection and quantitation of samples of human protein molecules.
2. Background Art
Protein molecules are produced by the cells of living organisms and are essential participants in most biological processes. Typically, each protein molecule in a living organism performs a specific function, such as facilitating a given metabolic activity or transporting chemical constituents. The specific function of a given protein molecule is determined by the sequence of amino acid building blocks that are connected in end-to-end relationship to make up the protein molecule. The precise sequence of the amino acids in each protein molecule is in turn a coded replication of a portion of the sequence of nucleotide building blocks in some gene in the genome of the organism in which the protein molecule has utility. A gene will thus embody a coded version of each protein molecule that corresponds thereto. Thus, the biological function of a protein molecule may be ascertained by studying the gene to which the protein molecule corresponds.
One process that can be used to determine the function of a protein molecule is the process of forward genetics. In forward genetics, the correlation of functions to protein molecules commences with the identification of a function occurring in an organism and proceeds to locate a protein that performs that function by making reference to the sequence of nucleotides in genes of the genome of the organism. The organism is subjected to conditions that cause the genome of the organism to change or mutate. Correspondingly, a mutant organism will result that is studied closely to identify functions present in the original organism that have been lost in the mutated organism. Correspondingly, the mutated genome is compared closely with the original genome to detect structural changes between the original genome and the mutated genome that can account for the observed loss of function between the original organism and the mutated organism. The protein encoded by any portion of the original genome not faithfully repeated in the mutated genome is then investigated as a candidate protein molecule that performs the function lost during the mutation process.
Although forward genetics can be used successfully to correlate functions to protein molecules in lower organisms, such as bacterial forward genetics is inappropriate for correlating function to protein molecules in humans, because humans cannot ethically be randomly mutated.
Instead, the contrasting process of reverse genetics is used to identify the functions of protein molecules in higher organisms. Reverse genetics is conducted in conjunction with proteomics, a study of protein molecules produced by cells in which the function of protein molecules is established by isolating and studying the protein molecules.
Reverse genetics commences with the known sequences of genes in the genome of a higher organism. Using known nucleotide sequence information for an individual gene, a protein encoded by the gene is produced. Then proteomics is used to determine the function of that protein molecule.
Although the function of individual protein molecules is determinable using reverse genetics, there are at least 30,000 genes in a human cell, and collectively these genes are estimated to be capable of producing between 300,000 and one million different proteins. One obstacle to using reverse genetics to rapidly establish the function of each protein molecule in a human or other higher organism, is that the determination of the function of a single human protein using proteomics yet requires substantial time.
Aberrant or mutant forms of protein molecules disrupt normal biological processes causing disease, including some cancers and inherited disorders, such as cystic fibrosis and hemophilia. Given sufficient time, it is hoped that the functions of the protein molecules produced by the cells of a healthy person can be established. Then any aberrant or mutant protein molecules not normally present in the cells of a healthy person can be detected. Abnormal protein molecules can then be used as markers indicating that cells are in a disease state.
One way that disease markers can be detected is by developing detector molecules that specifically bind, or attach, to given disease markers. To diagnose a patient for a disease, the blood of the patient is tested with a detector molecule corresponding to that disease. If the detector molecule does bind, the existence of the disease marker becomes apparent, and medical personnel can conclude that the specific disease to which the disease marker corresponds is present in the patient.
The identification of a disease marker associated with a given disease can yield new products that prevent, diagnose, or treat the corresponding disease. For example, detector molecules can be used to isolate given disease markers. Then the disease markers may be studied using proteomics.
One problem in the diagnosis of diseases in this manner is that some human proteins have not been characterized, and some diseases are as yet not diagnosable. Another drawback in diagnosing diseases in this manner is that some diseases produce only small numbers of disease markers in the blood of a victim, and thus cannot be visualized using known methods. Although detector molecules will bind to whatever corresponding disease markers are present in the blood, if the number of disease markers in a blood sample is few, known processes may not be sufficiently sensitive to permit those disease markers to even be detected.
Infections are caused by pathogenic microorganisms that invade the body of a patient. The pathogenic microorganisms produce virulence proteins, such as toxins, that then damage the tissues of the patient. Virulence proteins are, however, detectable in blood. Thus, the blood of a patient can be used to determine whether the patient is infected with a pathogenic microorganism. Detector molecules corresponding to specific virulence proteins are added to a blood sample. If detector molecules bind to a constituent of the sample, one or more of those specific virulence proteins are known to be in the sample, and the presence in the patient of an infection that produce virulence proteins is confirmed.
Since all virulence proteins of pathogenic microorganisms have not been identified, some infections are not diagnosable in this manner. Other infections are not diagnosable until late in the course of an infection, because the number of virulence proteins produced by the pathogenic microorganism early in the course of the infection is too small to be identified in the blood sample.
Two primary types of detector molecules are used to bind target analytes: antibodies and fusion molecules. Each type of detector molecule will be discussed individually. The term “target analyte” will be used herein to refer to the molecule that becomes bound by a given detector molecule. Examples of target analytes are disease markers, virulence proteins, nucleic acid molecules, protein sub-units, sugars, and lipids.
A first type of detector molecule used to bind target analytes is an antibody detector molecule. The antibody portion of the antibody detector molecule is a protein produced by the immune system of an animal in response to a target analyte foreign to the animal. Antibodies that bind specifically to the target analyte are generated by immunizing an animal with the target analyte itself. These antibodies bind to a specific site, or epitope, on the target analyte that was used to immunize the animal.
Antibodies produced by an animal in response to a target analyte are collected from the animal, and the antibodies are tagged with a detectable marker to form an antibody detector molecule.
Typically, the detectable marker is a chemical moiety that emits fluorescence, emits radioactivity, or exhibits enzymatic activity. The antibody detector molecule binds specifically to the target analytes that caused the antibody detector molecule to be produced. To determine whether the antibody detector molecule is bound to the target analyte, the presence of the detectable marker is sensed by searching for the fluorescence, the radioactivity, or the enzymatic activity that is reflective of the presence of the detectable marker by a given of the antibody detector molecule.
Although antibodies bind with a high specificity to the target analyte that caused the antibody to be produced, the immunization of an animal to generate antibodies, and the subsequent collection of the antibodies from the animal can take months to accomplish, representing a problem when the antibody detector molecules are needed in short order. Also, antibodies cannot be produced for some target analytes, because some target analytes do not generate an immune response in an animal.
Another disadvantage in using antibody detector molecules is that the detectable markers used to tag the antibodies are not capable of being amplified, or readily reproduced in a large number. Therefore, when a small number of antibody detector molecules bind to target analytes within a sample, the correspondingly small number of detectable markers in the sample cannot be detected, because the signal emitted by these correspondingly small number of detectable markers is too weak to be sensed by known processes. For instance, the sensor used to detect the emitted signal from the fluorescence, the radioactivity, or the enzymatic activity maybe present, but may not be sensitive enough to detect the weak signal.
Although the presence of target analytes in a sample can be detected using antibody detector molecules, the number of target analytes in a sample can only be estimated based on the relative amount of signal emitted from the detectable markers. The number of target analytes in the sample cannot be precisely counted, because the detector molecules of the antibody detector molecules are not capable of being amplified in a linear fashion.
A second type of detector molecule used to detect target analytes is a fusion molecule. A fusion molecule has a protein sub-unit linked to a ribonucleic acid molecule by a covalent bond. The protein sub-unit portion of the fusion molecule binds to the target analyte, and the ribonucleic acid molecule is used to announce the presence in a sample of a fusion molecule bound to that target analyte.
Ribonucleic acid molecules are not particularly stable. The environment within a cell contains numerous enzymes that degrade ribonucleic acid molecules. Therefore, in a study of proteins in a cell, enzymes from the environment of the cell degrade the ribonucleic acid portion of a fusion molecule of this type. Fusion molecules made up of a protein sub-unit linked to a ribonucleic acid molecule are thus not well suited for the study of proteins in living cells.
To studying a given protein molecule itself to ascertain the function of the given protein molecules, the function of the given protein molecules may be determined by identifying a target analyte that interacts with the given protein molecules. For example, a given protein molecule may bind to a deoxyribonucleic acid molecule of a gene in order to regulate expression of the gene. The function of the given protein molecule may thus be determined by identifying the gene that interacts with the given protein molecule.
The target analyte interacting with a given protein molecule in a cell may be determined by disrupting the cell to release the contents of the cell, including the given protein molecule and the target analyte. The contents of the cell are so treated as to link the given protein molecule to the target analyte, forming a complex. A detector molecule specific for the given protein molecule or the target analyte is used to isolate the complex. The target analyte in the complex is identified; and based on the identity of the target analyte, the function of the protein is inferred.
A complication exists in ascertaining the function of a given protein molecule in this manner, if the given protein molecule interacts non-specifically with multiple target analytes. If the given protein molecule non-specifically interacts with a random target analyte, the precise function of the given protein molecule will be improperly determined if the random target analyte bound thereto. Also, if the given protein does not interact with any target analyte in a cell, the biological function of the protein cannot be determined in this manner.
It is thus a broad object of the present invention to improve the processes used to study human proteins, thereby to improve molecular research and human healthcare.
It is also an object of the present invention to increase the speed and efficiency with which the function of human protein molecules can be determined.
It is a further object of the present invention to characterize unknown disease markers associated with diseases in humans.
An additional object of the present invention is to increase the sensitivity of detector molecules used to identify disease markers. A related object of the present invention is to identify unknown virulence proteins produced by pathogenic microorganisms.
Another object of the present invention is to decrease the amount of time required to produce detector molecules.
Yet another object of the present invention is to produce a detector molecule for target analytes that does not produce an immune response in an animal.
An additional object of the present invention is a detector molecule with an amplifiable detectable marker for more efficient detection that can be accurately quantified.
A further object of the present invention is an improved process for identifying a target analyte that interacts with a given protein molecule.
The present invention also has as an object a detector molecule that minimizes cross-linking and non-specific interactions with more than one target analyte.
Yet another object of the present invention is a detector fusion molecule that is stable for use in many environments. A related object of the present invention is thus a detector fusion molecule that will not be degraded by the environment of a cell.
To achieve the foregoing objects, and in accordance with the invention as embodied and broadly described herein, systems and methods are described for producing detector fusion molecules. Also provided are detector fusion molecules having detectable markers attached thereto.
In one aspect of the present invention, a method is provided incorporating teachings of the present invention that produces a fusion molecule useable as a detector fusion molecule for a predetermined target analyte. The method includes the step of attaching a reactive moiety to a first end of a protein sub-unit, thereby creating a reactive intermediate with the reactive moiety attached at a first end thereof. A coupling reagent is bonded to a first end of a nucleic acid molecule, forming a modified nucleic acid molecule. A reaction is catalyzed between the reactive moiety of the reactive intermediate and the coupling reagent of the modified nucleic acid molecule. The reaction displaces the reactive moiety from the first end of the reactive intermediate and forms a covalent bond between the first end of the reactive intermediate and the first end of the modified nucleic acid molecule.
One example of a coupling reagent that may be bonded to the first end of the nucleic acid molecule to form a modified nucleic acid molecule useful in the inventive method is a phosphoramidite-containing molecule. In another example, a cysteine-like moiety is attached to a nucleotide, thereby forming a modified nucleotide. The modified nucleotide is then linked to the first end of a nucleic acid molecule, thereby forming a modified nucleic acid molecule.
Examples of protein sub-units useful in the methods of the present invention include natural proteins, recombinant peptide aptamers, and synthetic peptides. Examples of nucleic acid molecules useful in the present invention comprise deoxyribonucleic acid molecules, double-stranded deoxyribonucleic acid molecules, ribonucleic acid molecules, and peptide nucleic acid molecules.
In another aspect of the present invention, a fusion molecule is described that binds to a predetermined target analyte. The fusion molecule includes a protein sub-unit, a linker attached to a first end of the protein sub-unit, and a deoxyribonucleic acid molecule attached at a first end thereof to the linker by a covalent bond.
Examples of linkers used in a fusion molecule incorporating teachings of the present invention include an amide linkage or a cysteine-like moiety attached to the carboxyl terminus of the protein sub-unit. The linker is covalently bonded to the 5′ end or the 3′ end of the deoxyribonucleic molecule by an amide bond.
In yet another aspect of the present invention, a detector fusion molecule comprises a protein sub-unit, a cysteine-like moiety attached to a first of the protein sub-unit, and a nucleic acid molecule attached at a first end thereof to the cysteine-like moiety by a covalent bond.
In one example, the cysteine-like moiety of the protein sub-unit comprises an amide linkage. Examples of nucleic acid molecules of the detector fusion molecule include deoxyribonucleic acid molecules, ribonucleic acid molecules, double-stranded deoxyribonucleic molecules, and peptide nucleic acid molecules.
Another aspect of the present invention includes a method for recognizing a target analyte in a sample. The method includes the step of manufacturing detector fusion molecules by attaching a quantity of reactive moieties to a first end of a quantity of protein sub-units, thereby creating a quantity of reactive intermediates with reactive moieties attached at a first end thereof. A quantity of coupling reagents is bonded to first ends of a quantity of nucleic acid molecules, thereby forming a quantity of modified nucleic acid molecules. A reaction is catalyzed between the reactive moieties of the reactive intermediates and the coupling reagents of the modified nucleic acid molecules. As a result of the reaction, the reactive moieties are displaced from the first end of the reactive intermediates, and covalent bonds are formed between the first end of the reactive intermediates and the first end of the modified nucleic acid molecules. The sample is contacted with a quantity of the detector fusion molecules, and the detector fusion molecules bind to target analytes in the sample. The nucleic acid molecules of the detector fusion molecules bound to the target analyte are then amplified, producing an amplification product. The amount of target analyte in the sample is quantified by determining the amount of the amplification product.
In one embodiment of the present invention, the nucleic acid molecule amplification is accomplished by hybridizing to the modified nucleic acid molecule of the bound detector fusion molecule a primer having a detectable marker and a nucleotide sequence complementary to a portion of a sequence of the modified nucleic acid molecule. A deoxyribonucleic acid polymerase is added to the bound detector fusion molecules. After amplification, the amount of the detectable marker is measured, thereby quantifying the amount of target analyte present in the sample.
In a further aspect of the present invention, a kit for recognizing or quantifying a target analyte is provided. The kit includes a detector fusion molecule comprising a protein sub-unit, a linker attached to a first end of the protein sub-unit, and a deoxyribonucleic molecule attached at a first end thereof to the linker by a covalent bond. The kit also includes a first means for amplifying the detector fusion molecule to produce an amplification product and a second means for visualizing the amplification product.
An example of such a first means is a deoxyribonucleic acid primer and a deoxyribonucleic acid polymerase. An example of such a second means is a detectable marker attached to the deoxyribonucleic acid primer.
Additional objects and advantages of the invention will be set forth in the description which follows and, in part, will be obvious from the description or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instruments and combinations pointed out in the appended claims.
In order that the manner in which the above-recited and other advantages and objects of the invention are obtained, a more particular description of the invention briefly described above will be rendered by reference to a specific embodiment thereof which is illustrated in the appended drawings. Understanding that these drawings depict only a typical embodiment of the invention and are not, therefore, to be considered limiting of its scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:
The following definitions are provided at the outset to facilitate the following descriptions. As used herein, the term “epitope” will be used to refer to a specific area, portion, or domain of a target analyte that associates, or binds, to a detector fusion molecule. The expression “mRNA” as used herein will be used to refer to messenger ribonucleic acid. As used herein, the expression “dNTP” will be used to refer to deoxynucleotide triphosphate. The expression “rNTPs” will be used herein to refer to ribonucleotide triphosphates. As used herein, the expression “DNA” will be used to refer to deoxyribonucleic acid, and the expression “RNA” will be used to refer to ribonucleic acid. The expression “PCR” will be used herein to refer to a polymerase chain reaction. As used herein, the expression “dUTP” will be used to refer to deoxyuracil triphosphate, the expression “dATP” will be used to refer to deoxyadenine triphosphate; the expression “dCTP” will be used to refer to deoxycytidine triphosphate; and the expression “dGTP” will be used to refer to deoxyguanosine triphosphate.
Protein sub-unit 22 in the generalized embodiment depicted in
Linker 24 of detector fusion molecule 20 of
Detector fusion molecule 20 of
Referring now to
Detector fusion molecule 46 includes a protein sub-unit 48 that is different from protein sub-unit 22 of detector fusion molecule 20. Protein sub-unit 48 has a binding site 50 that binds to an epitope such as, but possibly different from epitope 42 of target analyte 40 depicted in
Detector fusion molecule 54 includes a protein sub-unit 56 different from protein sub-unit 48 of detector fusion molecule 46 and different from protein sub-unit 22 of detector fusion molecule 20. Detector fusion molecule 54 includes nucleic acid molecule 60 that is shorter in length than nucleic acid molecules 26 and 52, and a binding site 58 specific for an epitope different from that of detector fusion molecules 20 and 46.
The three different detector fusion molecules 20, 46, and 54 illustrated in
Alternatively, nucleic acid molecules 26, 52, and 60 may have different nucleotide sequences. The amplification products produced from the nucleic acid molecules 26, 52, and 60 are distinguishable on the basis of sequence. In this manner, each nucleic acid molecule 26, 52, and 60 of the three detector fusion molecules 20, 46 and 54 serves as a barcode identifying each detector fusion molecule 20, 46, and 54. For example, a sequence of nucleic acid molecule 26 may be the sequence encoding protein sub-unit 22. Accordingly, detector fusion molecules 20, 46, and 54 may be used to identify a corresponding number of separate target analytes present simultaneously in a single sample.
In a step 82, reverse transcriptase and dNTPs are added to a solution containing protein sub-unit 22 attached to hybridized complex 76, thus reverse transcribing the DNA of hybridized complex 76 to RNA and forming a mature detector fusion molecule 20. Nucleic acid molecule 26 of the detector fusion molecule 20 of
Detector fusion molecule 20 is placed in contact with a sample 90 of target analytes at a step 83. Target analyte 40 with epitope 42 binds binding site 32 of detector fusion molecule 20. In a step 84, RNAse, an enzyme that degrades RNA is added to the solution containing detector fusion molecule 20 bound to target analyte 40. The RNAse degrades the ribonucleic acid molecule portion of nucleic acid molecule 26 and releases the DNA strand of nucleic acid molecule 26. Protein sub-unit 22 remains bound to target analyte 40 and to linker 24, which are separated from nucleic acid molecule 26. At a step 86, Klenow fragment, a DNA primer, and dNTPs are added to nucleic acid molecule 26, thereby allowing the Klenow fragment to polymerize the primer hybridized to the single-stranded DNA molecule and to form a double-stranded DNA molecule. At a step 88, T7 ribonucleic acid polymerase and rNTPs are added to produce amplification product 44.
Alternatively, at step a 88, Tax polymerase, primers complementary to each of the strands of the double-stranded DNA molecules, and dNTPs are added to the double-stranded DNA molecule, thereby producing an amplification product 44 that includes a plurality of nucleic acid molecules 26a through 26e. Whether amplification product 44 includes DNA or RNA as nucleic acid molecules 26a through 26e, amplification product 44 is detected using known processes to ascertain the presence of target analyte 40 in sample 90.
In
A protein sub-unit library may be obtained at dialog box 102 from a natural source by isolating a plurality of naturally occurring genes and expressing the naturally occurring genes in an expression vector, thereby producing a library of natural protein sub-units. The expressed natural protein sub-units are proteins that have not been genetically modified or mutated, but are obtained from genes in a wild type state and isolated from an organism.
Alternatively, the protein sub-unit library could be obtained at dialog box 102 from a recombinant source where genes encoding the protein sub-units are genetically modified, such as by fusing the gene, or portion of a gene, to another gene, thereby producing a recombinant gene. The recombinant gene is expressed in an expression vector, thereby producing a library of recombinant protein sub-units.
The protein sub-unit library may also be obtained at dialog box 102 from a synthetic source, wherein genes encoding the protein sub-unit are randomly synthesized, thereby producing a synthetic gene. The synthetic gene is expressed in an expression vector, thereby producing a library of synthetic protein sub-units. Alternatively, the synthetic protein sub-unit may be randomly synthesized using a protein synthesizer.
A target analyte is obtained at dialog box 104 from a natural, recombinant, or synthetic source. The target analyte may comprise any type of molecule, including without limitation, nucleic acid molecules, polypeptide molecules, protein molecules, polysacharrides, lipids, metals, minerals, vitamins, or any other type of known molecule. In a manner similar to obtaining the protein sub-unit library at dialog box 102, the target analyte may be obtained from a natural source, such as a target analyte that is isolated in an unmodified form, such that the natural target analyte represents the target analyte in a wild-type state.
Alternatively, the target analyte may be obtained at dialog box 104 from a recombinant source. For instance, the recombinant target analyte may be produced by fusing one natural target analyte to another target analyte, or the recombinant target analyte could be produced from or comprise a recombinant protein. The target analyte may also be obtained at dialog box 104 from a synthetic source. For example, the target analyte may be synthetically manufactured in vitro.
Once the protein sub-unit library and the target analyte are obtained at dialog boxes 102 or 104, a protein sub-unit that specifically binds the target analyte is isolated using known processes at dialog box 106, such as phage display, yeast two-hybrid, yeast display, bacterial display, bacterial two-hybrid, surface plasmon resonance, or any technique that allows for the specific interaction of two molecules to be determined. Once the protein sub-unit that specifically binds the target analyte is isolated at dialog box 106, the gene encoding for the protein sub-unit is isolated, and as illustrated in dialog box 108, the isolated protein sub-unit is attached to a reactive moiety, thereby producing a reactive intermediate.
Referring to dialog box 110, a sequence of a nucleic acid molecule used to produce a mature detector fusion molecule is determined. The sequence of the nucleic acid molecule may be a sequence of the gene encoding the protein sub-unit, a randomly determined sequence, or a sequence including, without limitation, a unique site, such as a restriction site on a promotion site, e.g., a T7 promoter sequence. The nucleic acid molecule used in the production of the mature detector fusion molecule may include any type of known nucleic acid molecule including DNA, RNA, a peptide nucleic acid (PNA) molecule, or any combination of nucleic acid molecules thereof. Regardless of the type of nucleic acid molecule selected, as depicted in dialog box 112, a coupling reagent is attached to the nucleic acid molecule, thereby forming a modified nucleic acid molecule. For instance, the detector fusion molecule can identify and quantify disease markers and virulence proteins as target analytes, thereby allowing medical practitioner 10 of
As indicated in dialog box 114, the reactive intermediate is placed in contact with the modified nucleic acid, such that a reaction is catalyzed between the reactive moiety of the reactive intermediate and the coupling reagent of the nucleic acid molecule, thereby forming the mature detector fusion molecule. As indicated in dialog box 116, the detector fusion molecule is used to identify and quantify a target analyte. For instance, the detector fusion molecule can identify and quantify disease markers and virulence proteins as target analytes, thereby allowing medical practitioner 10 of
As further illustrated in
At a step 142, an autocatalytic hydrolysis reaction is induced by adding a reactive moiety 144 and tris (2-carboxyethyl)-phosphine (TCEP) to a solution of the protein sub-unit-intein recombinant protein 130′. A pair of electrons from reactive moiety 144 attacks carbonyl carbon 136 of protein sub-unit intein recombinant protein 130′. At a step 146, intein molecule 132, or portion, attached to amino moiety 138 and thiol-containing group 134 are removed from protein sub-unit 22, which remains attached to reactive moiety 144, thereby forming a reactive intermediate 148. Reactive moiety 144 may comprise any thiol-containing group such as thiophenol or mercaptoethanesulfonic acid.
Lane 1 illustrates a protein size standard. Lane 4 represents the uninduced streptavidin-intein fusion sequence, and Lane 5 represents the induced streptavidin-intein fusion sequence. In Lane 5 a streptavidin-intein protein fusion is present at arrow 154, thus indicating that a streptavidin-intein recombinant protein of the expected site is produced.
Referring to Lanes 2 and 3, a gene coding for a peptide aptamer that binds human cyclin-dependent kinase 2 (hCDK2) is cloned and expressed as described herein with reference to the streptavidin-intein fusion sequence, thereby producing a peptide aptamer-intein fusion sequence. The peptide aptamer-intein fusion sequence is transformed and expressed in a bacterial cell. Lane 3 of
At a step 252, DNA polymerase, nucleotides, and a modified nucleotide 232 are added to symmetrical nucleic acid molecule 240 in such a manner that added nucleotides fill in nucleotide overhangs 250. In the illustrated embodiment, modified nucleotide 232 is cysteine-modified dUTP, and added nucleotides include DATP, dCTP, and dGTP. Since nucleotide overhangs 250 comprise the sequence TCGA, modified nucleotide 232 will be incorporated into symmetrical nucleic acid molecule 240 at each end complementary to the adenine A in each nucleotide overhang 250. The DATP, dCTP, and dGTP will be incorporated into symmetrical nucleic acid molecule 240, thereby forming a modified symmetrical nucleic acid molecule 256. At a step 254, a restriction enzyme is used to sever modified symmetrical nucleic acid molecule 256 at restriction enzyme site 248 into two modified nucleic acid molecules 186″ or fragments. Since restriction enzyme site 248 is located substantially equidistant from the pair of nucleotide overhangs 250, the resulting pair of modified nucleic acid molecules 186″ are substantially identical. Restriction enzyme site 248 maybe an EcoRI site, wherein the restricting enzyme EcoRI is used for severing modified symmetrical nucleic acid molecule 256.
In an alternative embodiment, a modified nucleotide maybe attached to a double-stranded nucleic acid molecule by nicking one end of the double-stranded nucleic acid molecule with a restriction enzyme, thereby producing a nicked double-stranded nucleic acid molecule with a terminal nucleotide removed from one end of the nicked double-stranded nucleic acid molecule. The modified nucleotide may be attached to the nicked double-stranded nucleic acid using a fill-in reaction, thereby creating a modified nucleic acid molecule.
At a step 260, reactive moiety 144 is displaced from reactive intermediate 148 by coupling reagent 160 of modified nucleic acid molecule 186, thereby forming linker 24 between protein sub-unit 22 and nucleic acid molecule 26. The displacement of reactive moiety 144 from protein sub-unit 22 results in the formation of a covalent bond between protein sub-unit 22 and coupling reagent 160 of modified nucleic acid molecule 186. An N—S acyl shift takes place at step 263, thereby producing a peptide bond 262 and resulting in mature detector fusion molecule 20. An amide bond 264 is also produced in linker 24 as a result of the N—S acyl shift, thereby resulting in linker 24 including an amide linkage.
Binding site 32 of protein sub-unit 22 of detector fusion molecule 20 specifically binds to epitope 42 of target analyte 40. At a step 274, any unbound detector fusion molecules 20 are washed away from the sample, thereby leaving only detector fusion molecules 20 that are specifically bound to target analytes 40. Nucleic acid molecule 26 of the detector fusion molecule 20 is amplified at step 276, thereby producing amplification product 44.
The type of nucleic acid molecule 26 used to produce detector fusion molecule 20 dictates, at least in part, a type of method used to produce the amplification product 44. For instance, if nucleic acid molecule 26 includes a T7 promoter sequence, then T7 polymerase and rNTPs could be used to amplify nucleic acid molecule 26. Alternatively, if nucleic acid molecule 26 comprises DNA, then PCR may be used to amplify nucleic acid molecule 26, wherein a primer complementary to a portion of the sequence of nucleic acid molecule 26 of detector fusion molecule 20 bound to target analyte 40 is hybridized to nucleic acid molecule 26. For PCR amplification, DNA-thermostable polymerase and dNTPs are added to produce amplification product 44. In the alternative, if nucleic acid molecule 26 of bound detector fusion molecule 20 is a hybrid RNA-DNA molecule, then RNAse maybe added, thereby releasing the single-stranded DNA molecule, which may be directly amplified using PCR as previously described herein. Alternatively, the single-stranded DNA molecule may be converted to double-stranded DNA using Klenow polymerase, or subsequently amplified using T7 RNA polymerase and rNTPs.
Amplification product 44 is identified by resolving amplification product 44 on a gel, such as agarose or polyacrylamide, and staining amplification product 44 with SYBR green or ethidium bromide. In yet another alternative embodiment, amplification product 44 may be identified by sequencing a nucleotide sequence of amplified nucleic acid molecules 26a through 26f using known processes.
To detect the ability of streptavidin detector fusion molecule to bind biotin, biotinylated-BSA or BSA alone are used as target analytes. Since streptavidin is known to bind specifically to biotin, streptavidin detector fusion molecule works as an example to illustrate a detector fusion molecule produced using the methods of the present invention binding a target analyte. Biotinylated-BSA or BSA alone is non-specifically adsorbed to wells of a polystyrene ELISA plate, and surfaces of ELISA plate not adsorbed with biotinlyated-BSA or BSA alone are blocked with 0.2% casein. Streptavidin detector fusion molecule is added to loaded wells, thus allowing streptavidin detector fusion molecules to bind biotin adsorbed to wells of the ELISA plate. Unbound streptavidin detector fusions are removed from wells by extensive washing.
The presence of bound streptavidin detector fusion molecules is detected by adding T7 polymerase, rNTPs, and buffer to the wells to amplify nucleic acid molecules of bound streptavidin detector fusion molecules. RNA synthesis is conducted for four hours. Amplification products of the RNA synthesis are fractionated on a 10% acrylamide, 50% urea denaturing gel 288, and stained with SYBR green II, an agent used to stain single-stranded RNA, thereby illuminating amplification products. Amplification products are illustrated in
Negative controls are present in each of Lane 1 that lacks biotin-BSA, in Lane 2 that lacks T7 polymerase, and in Lane 3 that lacks the streptavidin detector fusion. As seen in Lanes 1, 2, and 3, no significant amplification product was generated. Lane 5 includes T7 polymerase and streptavidin detector fusions, and Lane 6 is a positive control including T7 polymerase, a double-stranded DNA sequence including a T7 promoter sequence, and BSA. As shown in Lanes 5 and 6, an RNA amplification product is generated. Lane 4 includes biotinylated-BSA, T7 polymerase, and streptavidin detector fusion molecules. After amplification, Lane 4 is depicted as producing an amplification product, thus demonstrating that streptavidin detector fusion molecules bind specifically to biotin, and that nucleic acid molecules of streptavidin detector fusion molecules are amplifiable.
An immobilized sample 302 including an individual magnetic micro-particle 304 and a number of individual sample molecules, or target analytes 306a through 306d, are illustrated in
At a step 318, nucleotides and a polymerase, such as a DNA or an RNA polymerase, are added to bound detector fusion molecules 312a through 312d, such that polymerase will amplify nucleic acid molecule 26 of bound detector fusion molecules 312a through 312d, thereby producing amplification products 320a through 320d for bound detector fusion molecules 312a through 312d. Amplification products 320a through 320d are identified or quantified using various known methods. Identification of the amplification products 320a through 320d allows medical practitioner 10 of
In a first method, amplification products 320a though 320d are passed through a HPLC column 352, such that amplification products 320a through 320d may be separated on a basis of chemical composition.
In a second method indicated at a step 324, amplification products 320a through 320d are processed with quantitative PCR 354 using a plurality of detection techniques, such that after a specified number of rounds of PCR, PCR products and quantitated by measuring an amount of radioactivity or fluorescence emitted by detectable markers. Other detectable markers that may be used include intercalating fluorescent dyes, such as Hoescht 33342, that are detectable with fluorescence microscopes.
In a third method displayed at a step 326, amplification products 320a through 320d are placed on a hybridization microarray 356 or chip. As depicted in
An example of a method used to detect an amplification product produced by amplifying a nucleic acid molecule of a detector fusion molecule is illustrated in
A capillary electrophoresis chromatogram 340 is generated, and peaks 342a through 342c of capillary electrophoresis chromatogram 340 indicate the presence of hybridized duplexes 336a through 336c. In this embodiment, detector nucleic acid molecules 334a through 334c may be used to distinguish single-stranded nucleic acid molecules 330a through 330d on a basis of sequence because the single-stranded overhangs 344a through 344c of hybridized duplexes 336a through 336c each have a different nucleotide sequence. The different nucleotide sequences of single-stranded overhangs 344a through 344c are designed to specifically hybridize to a sequence of each of the single-stranded nucleic acid molecules 330a through 330c. In this manner, since hybridized duplexes 336a through 336c are of different lengths, peaks 342a through 342c of capillary electrophoresis chromatogram 340 are used to distinguish each of single-stranded nucleic acid molecules 330a through 330c on a basis of size.
In another embodiment, amplification products 320a through 320d produced with a primer labeled with a detectable marker are resolved on a gel, thereby forming bands of amplification products 320a through 320d separated on a basis of size. An intensity of bands of amplification products 320a through 320d is measured using known processes, such as using a phosphoimager to measure radioactivity emitted by detectable markers of primers.
As an example of how detector fusion molecules of the present invention are used to quantify an amount of a target analyte, reference is made to
T7 RNA polymerase and rNTPs are added to ELISA plate wells, thereby amplifying nucleic acid molecules of detector fusion molecules bound to the adsorbed hCDK2 target analytes, thereby producing an amplification product. Amplification product is resolved on a denaturing PAGE gel of 10% acrylamide and 50% urea and stained with SYBR green II. A photograph 368 of the resolved amplification product is depicted in
An amount of the amplification product observed on photograph 368 of
Referring in conjunction to
Fusion molecule-analyte complexes 388a through 388c are depicted in
Detector fusion molecules of the present invention are illustrated in
Detector fusion molecules of first tube 412 are of the type of detector fusion molecules 20 described herein with reference to
In an alternative embodiment, second tube 414 may comprise a DNA polymerase and a DNA primer. DNA primer may have a detectable marker attached thereto. In this alternative embodiment, fourth tube 418 containing dNPTs will be included in kit 410. DNA polymerases that are used include, without limitation, Klenow fragment, Tax polymerase, Vent polymerase, and Deep Vent polymerase.
In any of kit 410 embodiments, second means of third tube 416 includes a stain for visualizing the amplification product. Stains that may be used include ethidium bromide and SYBR green II. In an alternative embodiment, a fifth tube 420 containing a buffer solution for providing optimal binding conditions of detectable fusion molecule marker to target analytes are included.
The invention maybe embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
This application is a continuation-in-part application of U.S. Provisional Patent Application Ser. No. 60/374,795 that was filed on Apr. 23, 2002.
| Number | Date | Country | |
|---|---|---|---|
| 60374795 | Apr 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10218233 | Aug 2002 | US |
| Child | 11286057 | Nov 2005 | US |
