Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-22
2004-11-02
Forman, BJ (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S283100, C435S287100, C435S287200, C435S288300, C435S288700, C436S501000, C422S050000, C422S068100, C536S023100, C536S024100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06811973
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to array based assays and more particularly to microarrays or beads having labeled probes molecules which are designed to assist in the identification and quantification of target molecules including native molecules or mutant molecules.
BACKGROUND OF THE INVENTION
Currently, scientists are interested in examining the effect of different treatments or conditions on the human body. One method of exploring these effects is to examine the changes that occur in the expression or function of molecules within individual cells or tissues which are exposed to the treatments or conditions. The molecules which are effected by such exposure may then be qualitatively or quantitatively compared to the native molecules to examine the effect of the treatment or condition.
In determining the expression or function of any molecule, traditional methods in molecular biology are only useful at examining the effect of one agent on one cellular molecule, in one experiment, which means that any effect of on any given molecule, in general, is slow, expensive and difficult to assess. The advent of microarray technology has allowed scientists to examine the effect of one treatment or condition or combination of thereof on thousands of molecules simultaneously.
Microarray technology consists, generally, of probe molecules being attached to a solid substrate and target molecules, obtained from the exposed cells contacting the probe molecules. Typically, target molecules are labeled prior to exposure to the microarray. Once exposed to the microarray, some target molecules selectively form probe/target pairs by binding/hybridizing with the complimentary probe molecules on the microarray. The target molecules that do not form pairs are removed from the microarray. Where the probe/target pairs are formed on the microarray, the scientist can then visualize the probe molecules which were bound by labeled target molecules. The relative amount of probe/target pairs which form can be compared between groups of cells which are exposed to different treatments and cells which are not (controls) to determine the effect of the treatment. For example, the levels of expression of mRNA or protein as a target molecule may have changed, or alternatively, the conformation of a protein or carbohydrate may have changed. As thousands of molecules can be screened simultaneously using this technology, microarrys may be used to improve timeliness, effectiveness, accuracy and overall benefit-to-cost ratio for examining changes in molecular expression and function relative to traditional methods.
One difficulty in microarray technology thus far, however, has been the ability of scientists to efficiently and effectively identify and quantify the probe/target pairs which form on the microarray. As above, the target molecule is typically labeled, and that label is detected to identify the probe/target pair. However, the label may not be present on the target molecule in sufficient amounts to be detectable. If a target sequence is not adequately labeled, false negative results are obtained, meaning that probe/target pairs are formed, but not identified by the scientist. Labeling inadequacies often occur due to enzymatic reproducibility, inhibition and or incomplete incorporation of dyes.
SUMMARY OF THE INVENTION
Labeled probe molecules are disclosed where the label is detectable when the probe molecule is not paired with a complimentary target molecule, and the label is undetectable when paired with a target molecule. Such a system allows for a means of identifying target molecules in a sample without encountering the difficulty of labeling target molecules.
The probe and target molecules can be polymers of nucleic acids, amino acids or carbohydrates. The label is preferably fluorescent, and can be detected by those methods currently known or to be developed in the art, such as flow cytometry. The probe molecules can be attached to a solid substrate such as a microarray or a bead.
In one embodiment, the probe molecules are single-stranded, fluorescently labeled nucleotide sequence which are attached to a microarray. The fluorescing nucleotide probe molecule is quenched when hybridized to a complimentary target nucleotide sequence.
In an alternate embodiment, the probe molecules are single-stranded, fluorescently labeled oligonucleotide sequences comprised in part or entirely by nucleotide base analogs.
Such a system also allows for a means of quantifying target molecules in a sample. In an alternate embodiment, a known number of labeled probe molecules are attached to a bead which can be contacted with the target sequences within a cellular sample. When probe/target pairs are formed, the fluorescence of the probe molecule will be decreased. When a number of probe/target pairs are formed which is substantially equal to the number of probe molecules present on the head, the fluorescence of the probe molecule will be substantially eliminated. Thus, the beads can be used to quantify the amount of a target molecule in a cellular sample.
The above described and many other features and attendant advantages of the present invention will become apparent from a consideration of the following detailed description when considered in conjunction with the accompanying drawings.
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Berman, Esq. Charles
Forman BJ
Greenberg & Traurig, LLP
The Regents of the University of California
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