Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2001-10-24
2004-11-09
Riley, Jezia (Department: 1637)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C435S006120, C530S300000, C530S350000, C514S023000, C536S017200, C560S001000, C560S008000
Reexamination Certificate
active
06815212
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to methods and compositions for analyzing nucleic acid molecules, and more specifically, to the use of specialized tags and linkers which may be utilized to enhance sensitivity of the analysis of a wide variety of biological-based assays.
BACKGROUND OF THE INVENTION
Detection and analysis of nucleic acid molecules are among the most important techniques in biology. They are at the heart of molecular biology and play a rapidly expanding role in the rest of biology.
Generally, following essentially all biochemical reactions, analysis entails some form of detection step. Of especial concern is the detection of nucleic acid hybridizations and antibody-antigen binding. Ideally, detection should be sensitive and allow processing of multiple samples. However, current detection techniques are somewhat limited in both these characteristics.
Hybridization of nucleic acid molecules is generally detected by autoradiography or phosphor image analysis when the hybridization probe contains a radioactive label or by densitometer when the hybridization probe contains a label, such as biotin or digoxin, that is recognized by an enzyme-coupled antibody or ligand. When a radiolabeled probe is used, detection by autoradiography suffers from film limitations, such as reciprocity failure and non-linearity. These film limitations can be overcome by detecting the label by phosphor image analysis. However, radiolabels have safety requirements, increasing resource utilization and necessitating specialized equipment and personnel training. For such reasons, the use of nonradioactive labels has been increasing in popularity. In such systems, nucleotides contain a label, such as biotin or digoxin, which can be detected by an antibody or other molecule that is labeled with an enzyme reactive with a chromogenic substrate. Alternatively, fluorescent labels may be used. These systems do not have the safety concerns as described above, but use components that are often labile and may yield nonspecific reactions, resulting in high background (i.e., low signal-to-noise ratio).
Antibody-antigen binding reactions may be detected by one of several procedures. As for nucleic acid hybridization, a label, radioactive or nonradioactive, is typically conjugated to the antibody. The types of labels are similar: enzyme reacting with a chromogenic substrate, fluorescent, hapten that is detected by a ligand or another antibody, and the like. As in detection of nucleic acid hybridization, similar limitations are inherent in these detection methods.
The present invention provides novel compositions which may be utilized in a wide variety of nucleic acid-based, or protein (e.g., antibody)—based procedures, and further provides other, related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods which may be utilized to enhance sensitivity and sample number throughput in a wide variety of based assays. In particular, based upon the inventions described herein, many assays that heretofore have taken a long period of time to complete may now be performed ten to more than a hundred-fold faster. The methods described herein thus represent a dramatic and important improvement over previously available assays.
For example, within one aspect of the invention methods are provided for detecting the binding of a first member to a second member of a ligand pair, comprising the steps of (a) combining a set of first tagged members with a biological sample which may contain one or more second members, under conditions, and for a time sufficient to permit binding of a first member to a second member, wherein said tag is correlative with a particular first member and detectable by non-fluorescent spectrometry, or potentiometry, (b) separating bound first and second members from unbound members, (c) cleaving the tag from the tagged first member, and (d) detecting the tag by non-fluorescent spectrometry, or potentiometry, and therefrom detecting the binding of the first member to the second member.
A wide variety of first and second member pairs may be utilized within the context of the present invention, including for example, nucleic acid molecules (e.g., DNA, RNA, nucleic acid analogues such as PNA, or any combination of these), proteins or polypeptides (e.g., an antibody or antibody fragment (e.g., monoclonal antibody, polyclonal antibody, or a binding partner such as a CDR), oligosaccharides, hormones, organic molecules and other substrates (e.g., xenobiotics such as glucuronidase—drug molecule), or any other ligand pair. Within various embodiments of the invention, the first and second members may be the same type of molecule or of different types. For example, representative first member second member ligand pairs include: nucleic acid molecule
ucleic acid molecule; antibody
ucleic acid molecule; antibody/hormone; antibody/xenobiotic; and antibody/protein.
Preferably, the first member will recognize either a selected second member specifically (i.e, to the exclusion of other related molecules), or a class of related second member molecules (e.g., a class of related receptors). Preferably the first member will bind to the second member with an affinity of at least about 10
−5
/M, and preferably 10
−6
/M, 10
−7
/M, 10
−8
/M, 10
−9
/M, or greater than 10
−12
/M. The affinity of a first molecule for a second molecule can be readily determined by one of ordinary skill in the art (see Scatchard,
Ann. N.Y. Acad. Sci
. 51:660-672, 1949).
Within other related aspects of the invention, methods are provided for analyzing the pattern of gene expression from a selected biological sample, comprising the steps of (a) exposing nucleic acids from a biological sample, (b) combining the exposed nucleic acids with one or more selected tagged nucleic acid probes, under conditions and for a time sufficient for said probes to hybridize to said nucleic acids, wherein the tag is correlative with a particular nucleic acid probe and detectable by non-fluorescent spectrometry, or potentiometry, (c) separating hybridized probes from unhybridized probes, (d) cleaving the tag from the tagged fragment, and (e) detecting the tag by non-fluorescent spectrometry, or potentiometry, and therefrom determining the patter of gene expression of the biological sample. Within one embodiment, the biological sample may be stimulated with a selected molecule prior to the step of exposing the nucleic acids. Representative examples of “stimulants” include nucleic acid molecules, recombinant gene delivery vehicles, organic molecules, hormones, proteins, inflammatory factors, cytokines, drugs, drug candidates, paracrine and autocrine factors, and the like.
Within the context of the present invention it should be understood that “biological samples” include not only samples obtained from living organisms (e.g., mammals, fish, bacteria, parasites, viruses, fungi and the like) or from the environment (e.g., air, water or solid samples), but biological materials which may be artificially or synthetically produced (e.g., phage libraries, organic molecule libraries, pools of genomic clones and the like). Representative examples of biological samples include biological fluids (e.g., blood, semen, cerebral spinal fluid, urine), biological cells (e.g., stem cells, B or T cells, liver cells, fibroblasts and the like), and biological tissues.
Within various embodiments of the above-described methods, the nucleic acid probes and or molecules of the present invention may be generated by, for example, a ligation, cleavage or extension (e.g., PCR) reaction. Within other related aspects the nucleic acid probes or molecules may be tagged at their 5′-end, and the so-tagged molecules function as oligonucleotide primers or dideoxynucleotide terminators.
Within other embodiments of the invention, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 , 70, 80, 90, 100, 200, 250, 300, 350, 400, 450, or greater than 500 different and unique tagged molecules may be ut
Howbert J. Jeffry
Mulligan John T.
Ness Jeffrey Van
Tabone John C.
Qiagen Genomics, Inc.
Riley Jezia
Seed Intellectual Property Law Group PLLC
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