Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-08-06
2002-04-02
Kunz, Gary L. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C435S006120, C435S975000
Reexamination Certificate
active
06365731
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular and cellular biology. More particularly, it concerns compositions and methods for removal of nucleic acid probes from sample nucleic acids, for example, sample nucleic acids attached to a solid support. The invention also concerns methods of stripping and reusing nucleic acid blots.
2. Description of Related Art
The central dogma of molecular biology holds that genetic information flows from nucleic acids (DNA and RNA) to proteins, and that the functions and relative abundances of proteins within a biological system defines that system. Proteins are polymers of amino acids and it is the sequence of amino acids within the polymer that determines the function of the protein. Amino acid sequences are encoded by nucleic acid sequences that exist in domains known as genes that are present in the genomes of biological entities. Genomes are the nucleic acid storehouses of genetic information present in all known living systems.
Genomes are typically composed of DNA, though there are a number of viruses with RNA genomes. The genes in DNA are converted to proteins via an RNA intermediate known as messenger RNA (mRNA). The rate of conversion of a DNA segment composing a gene to mRNA, and the subsequent degradation rate of that mRNA, affects protein production which ultimately impacts the state of that organism. The importance of nucleic acids in biology is unquestioned, thus a substantial amount of research effort has been applied to identifying gene sequence, structure, and function, as well as the relative rates of messenger RNA synthesis and degradation and the abundance of particular mRNA species.
A variety of techniques have been employed in the detection and quantification of both RNA and DNA. Most of these techniques rely on the capacity of nucleic acids to interact in a sequence specific manner known as hybridization. Hybridization occurs when two nucleic acid molecules possessing complementary sequences interact to form a, typically, double helical structure. Hybridization studies designed to identify or quantify a sample nucleic acid possessing a given sequence typically involve synthesizing an isotopically or non-isotopically labeled nucleic acid probe with a sequence that is complementary to the sample nucleic acid to be detected. The labeled nucleic acid probe is incubated with the sample population to allow hybridization between target and probe. Labeled nucleic acids that have not hybridized are removed, leaving the hybridized probe to be detected via the isotopic or non-isotopic label.
Of the many techniques for nucleic acid analysis that rely on hybridization, some involve immobilizing the sample nucleic acid on a solid support. Detection and/or quantification methods that rely on immobilization of RNA or DNA species by physical attachment to a solid support are well-known and include, but are not limited to, Northern (RNA sample nucleic acid), Southern (DNA sample nucleic acid), dot, slot, colony lifts, and related blot analyses (Maniatis et al., 1989), sandwich hybridization (Kwoh et al., 1989), and hybridization of a labeled amplification product to an oligonucleotide attached to a solid support (Woolford and Dale 1992).
In blot analyses, the solid support is usually a membrane and the sample is usually a heterogeneous population of nucleic acids purified from a cell culture, tissue, or organism. UV or chemically induced crosslinking between the sample and membrane generates the sample matrix. Isotopically or non-isotopically labeled probes (nucleic acids possessing sequences complementary to the RNA or DNA to be detected) are synthesized by enzymatic or synthetic means and mixed with the sample matrix. Hybridization of the probe to the subset of nucleic acids with complementary sequences, removal of the non-hybridized probe molecules by extensive washing, and detection of the remaining label provides for the positive identification and quantification of nucleic acids possessing the given sequence.
The sample matrices can be used multiple times, provided that all hybridized probe is removed prior to initiating hybridization of a new probe. The complete removal of the preceding probe is important, as the signal from one probe can affect the detection and quantification of a second nucleic acid. Removing any hybridized nucleic acid requires that sufficient energy be introduced to disrupt all hydrogen bonding and stacking interactions between probe and target. Additionally, the solution must be stringent enough to deny reannealing once the interaction between probe and target has been disrupted. Temperatures that exceed the melting temperature of the probe and target in solutions that lack monovalent salts and include detergents are typically used to remove probes from a solid support (Maniatis et al., 1989). However, in many instances, this level of stringency is inadequate to completely remove the probe leaving residual signal that can affect the subsequent analysis of other targets. In addition, the extreme conditions often cause irreversible damage to the sample matrix, either by removing sample nucleic acids altogether, or altering their chemical properties. These two problems often necessitate that a sample matrix be used one time and then discarded.
Given the time and expense involved in the preparation of a sample matrix, in addition to the fact that some sample matrices would be difficult to reproduce given the unavailability of starting materials (for example, patient samples, etc.), the ability to reuse a sample matrix would represent a significant advance in the art.
SUMMARY OF THE INVENTION
The present invention overcomes the limitations present in the art by providing compositions and methods for removing hybridized nucleic acid probes from sample nucleic acids, for example from sample matrices consisting of sample nucleic acids attached to solid supports. The invention provides for the specific degradation or cleavage of a nucleic acid probe following hybridization and detection, reducing the energy of hybridization between the sample nucleic acid and the various nucleic acid probe molecules. The reduction in hybridization energy provides for complete removal of the nucleic acid probe under conditions that are less likely to affect the sample nucleic acids and/or sample matrix (lower temperature and/or less extreme conditions). The present invention thus provides for the reuse, and even multiple uses, of sample matrices. The present invention also provides kits, including, but not limited to, kits for nucleic acid probe degradation, cleavable nucleic acid probe synthesis, sample matrix stripping and reuse, and nucleic acid detection.
For clarity, the nucleic acid(s) attached to a solid support will be referred to as the sample nucleic acid(s) or target; the solid support plus sample nucleic acid(s) will be referred to as the sample matrix; and the nucleic acid probe (or “probe”) will be the nucleic acid molecule that hybridizes to the sample nucleic acid(s).
Various basic aspects of the invention are summarized as follows. Note that, in accordance with long-standing patent law practice and convention, the words “a” and “an” denote “one or more” when used in this application, including the claims. Further, the breaking of a first bond in a nucleic acid probe can occur in conjunction with the breaking of other bonds, and there is no limitation in the invention as to breaking of “one and only one bond.”
The invention provides a method of removing a nucleic acid probe from a sample nucleic acid comprising obtaining a sample nucleic acid and a nucleic acid probe, the nucleic acid probe associated with the sample nucleic acid, breaking at least a first bond of the nucleic acid probe, and removing the nucleic acid probe from the sample nucleic acid. Of course, more bonds than a first bond may be broken, and, typically, more than one bond will be broken.
In certain embodiments, the sample nucleic acid probe comprises DNA. In other embodiments,
Brown David
Winkler Matthew
Ambion Inc.
Fulbright & Jaworski L.L.P.
Kunz Gary L.
Owens Howard V.
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