Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-09
2003-11-18
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S287200, C536S023100, C536S024300, C536S025400, C536S026600
Reexamination Certificate
active
06649351
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to the field of genome and proteome analysis and, more specifically to methods for detecting multiple analytes using mass spectrometry.
Molecular assays have been developed that can identify and quantitate a single analyte, such as a nucleic acid or protein, in a biological sample. These assays can be used, for example, to detect a known mutation in a gene, an infectious agent, or a protein associated with a disease such as cancer. The need to identify and quantitate many analytes from the same sample has become increasingly apparent in many branches of medicine. For example, it can be desirable to analyze a single sample for the presence of several infectious agents at once, for several genes that are involved in a particular disease, or for several genes that are involved in different diseases.
The full sequencing of the human genome has facilitated methods for comparing all of the genes between different cells or individuals. Different individuals are known to contain single base pair changes, called single nucleotide polymorphisms (SNPs), throughout their genomes. It is believed that there will be about one polymorphism per 1,000 bases, resulting in a large number of differences between individuals. These single nucleotide differences between individuals can result in a wide variety of physiological consequences. For example, the presence of different SNPs in cytochrome P450 genes can predict the ability or inability to metabolize certain drugs. Screening individuals for the presence of multiple SNPs could be used to predict how an individual will respond to a particular drug or treatment.
DNA microarrays are devices that contain thousands of immobilized DNA sequences on a miniaturized surface. Arrays have made the process of detecting several genes from a single sample more efficient. Unfortunately, despite the miniaturization of microarray formats, this method still requires significant amounts of the biological sample. In addition, in microarray methods there is a trade-off between high dynamic range and high sensitivity so that in order to increase dynamic range to detect genes of various abundance levels, there is a concomitant decrease in sensitivity.
Proteomics is the study of proteins expressed in a cell. Although more complex than genomics, proteomic analysis can give a more accurate picture of the state of a cell than genomic analysis. For example, the level of mRNA transcribed from a gene does not always correlate to the level of expressed protein. Therefore, analysis of gene expression alone does not always give an accurate picture of the amount of protein derived from a gene of interest. In addition, many proteins are post-translationally modified and these modifications are often important for activity. The type and level of modification of a protein can not be accurately predicted using genome analysis. Therefore, it is important to study a cell in terms of the proteins that are present. For example, it can be desirable to identify and quantitate all proteins present in a cell from an individual and compare the profile with other cells from the same or different individuals.
Assays for the detection of single proteins using antibody-based assays are available. However, analysis of several proteins simultaneously in the same sample can be more difficult. Two-dimensional gel electrophoresis has been used to study the protein content of a cell. This technique requires an individual gel for each sample and sophisticated software to compare the pattern of protein spots between gels. In addition, it is difficult to detect low abundance proteins using this method and several proteins, such as membrane proteins or proteins of very low or high molecular weight, are not ameable to the analysis.
Another aspect of proteome analysis is the study of protein-protein interactions within a cell. These protein-protein interactions form the basis of biochemical pathways within the cell. Two-hybrid assays have been used to study individual protein-protein interactions. However, this assay requires the cloning of the gene for a protein of interest into expression vectors, which is a labor-intensive process. In addition, two-hybrid assays often have a high rate of false positives where the protein of interest non-specifically interacts with another protein. Furthermore, two hybrid assays require several days to perform due to the growth cycle of the cells explored, which limits the number of assays can be performed at one time.
Thus, there exists a need for methods to identify and quantitate a plurality of analytes, including nucleic acids and proteins, quickly and with high sensitivity, high accuracy, and a large dynamic range. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The invention provides a method for detecting a target nucleic acid sequence, by: (a) contacting one or more target nucleic acid sequences with a set of tagged probes under conditions sufficient for hybridization of a target nucleic acid sequence with a tagged probe, the tagged probes containing a mass modifier region attached to a nucleic acid target binding moiety by a bond that is cleavable by a nuclease, the nucleic acid target binding moiety containing at least one bond resistant to the nuclease; (b) treating the tagged probe hybridized to the target nucleic acid with a nuclease under conditions sufficient for cleavage of the nuclease-cleavable bond to release a tag reporter, and (c) detecting a mass of the tag reporter, the mass uniquely corresponding to a known target sequence.
The invention also provides a method for detecting a target analyte, by: (a) contacting one or more target analytes with a set of tagged probes attached to a cleavage-inducing moiety under conditions sufficient for binding of a target analyte with a tagged probe, the tagged probes containing a mass modifier region attached to a target binding moiety by a cleavable linkage, the cleavable linkage being susceptible to cleavage when the cleavage-inducing moiety is activated by visible light; (b) separating tagged probes bound to a target binding moiety from unbound tagged probes; (c) activating the cleavage-inducing moiety with visible light to release a tag reporter, and (d) detecting a mass of the tag reporter, the mass uniquely corresponding to a known target analyte.
The invention further provides a method for detecting a target analyte, by: (a) contacting one or more target analytes with a set of first and second binding reagents under conditions sufficient for binding of a target analyte with the first and second binding reagents, each of the first binding reagents containing a cleavage-inducing moiety and a target binding moiety, each of the second binding reagents containing a tagged probe having a mass modifier region attached to a target binding moiety by a cleavable linkage, the cleavable linkage being susceptible to cleavage when in proximity to an activated cleavage-inducing moiety; (b) activating the cleavage-inducing moiety to release a tag reporter, and (c) detecting a mass of the tag reporter, the mass uniquely corresponding to a known target analyte.
The invention also provides a method for identifying a binding partner of a specific binding pair, by: (a) incorporating a cleavage-inducing moiety into a first binding partner of a specific binding pair; (b) contacting the first binding partner having an incorporated cleavage-inducing moiety with a set of second binding partners under conditions sufficient for binding, each of the second binding partners containing a tagged probe having a mass modifier region attached to a target binding moiety by a cleavable linkage, the cleavable linkage being susceptible to cleavage when in proximity to an activated cleavage-inducing moiety; (c) activating the cleavage-inducing moiety to release a tag reporter, and(d) detecting a mass of the tag reporter, the mass uniquely corresponding to a known second binding partner of a specific binding pair.
REFERENCES:
Chenna Ahmed
Hernandez Vincent S.
Hooper Herbert
Matray Tracy J.
Singh Sharat
Aclara Biosciences, Inc.
Macevicz Stephen C.
Tung J.
Whisenant Ethan
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