Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-07-13
2004-12-07
Sisson, Bradley L. (Department: 1634)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C536S025320
Reexamination Certificate
active
06828142
ABSTRACT:
FIELD OF INVENTION
This invention relates to novel compositions comprising functionalized nanocrystals. More particularly, the present invention relates to water-soluble nanocrystals which further comprise strands of polynucleotides of a known sequence, and the use of such functionalized nanocrystals to provide signal and signal amplification for detecting target molecules.
BACKGROUND OF THE INVENTION
Nonisotopic detection systems have become a preferred mode in scientific research and clinical diagnostics for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, immunoassays, histo-chemistry, and functional assays involving living cells. In particular, while fluorescent organic molecules such as fluorescein and phycoerythrin are used frequently in detection systems, there are disadvantages in using these molecules in combination. For example, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule. However, even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent have limited fluorescence intensity. Further, currently available nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules which can be used to label a biomolecule to be detected.
Branched DNA or DNA dendrimers have been constructed as a signal amplification tool (see, e.g., U.S. Pat. Nos. 5,487,973, 5,484,904, and 5,175,270). These matrices are comprised of a DNA backbone having DNA arms. For example, matrices are constructed of successive subunits of a double-stranded DNA with single stranded arms on each end. Some of the arms are used to hybridize to a specific oligonucleotide probe, whereas other arms are used to bind to a nonisotopic or isotopic label. While providing for the addition of an relative increase in the number of label molecules as compared to other systems, one disadvantage is that a subunit needs to be custom-synthesized to contain at least one arm consisting of a complementary sequence which is capable of hybridizing to the specific DNA sequence which the user wishes to detect. Additionally, the label molecules are attached or ligated to the outwardly extending ends (tips) of the DNA matrices, rather than as an integral part of the matrix.
Thus, there remains a need for a nonisotopic detection system which (a) can result in generation of a signal comprising fluorescence emission of high quantum yield; (b) can result in signal amplification; (c) is not limited as to the chemical nature of the target molecule to be detected (e.g., versus detection of nucleic acid molecules only); (d) can be used to bind molecular probes of various types (versus binding to oligonucleotide probes only); (e) is preferably universal in terms of detecting target molecules of various sequences; and (f) can result in the simultaneous detection of more than one type of target molecule by utilizing a class of nonisotopic molecules that may be excited with a single excitation light source and with resultant fluorescence emissions with discrete fluorescence peaks.
SUMMARY OF THE INVENTION
The present invention provides methods, compositions, and kits for use in an amplifiable, non-isotopic detection systems. The composition comprises nanocrystals that are functionalized to be water-soluble, and further functionalized to comprise a plurality of polynucleotide strands of a known (predetermined) sequence which extend outwardly from each nanocrystal. While there are several variations of this system, a basic principle of the invention is that a molecular probe is used to detect a target molecule, if present in a sample, by the binding specificity of the molecular probe for the target molecule or a portion thereof; and generation and amplification of a detectable signal by using at least two species of functionalized nanocrystals. A first species of functionalized nanocrystals (“primary dots”) have extending therefrom strands of polynucleotides of known sequence, and wherein the primary dots are, or become, operably linked to the molecular probe. A second species of functionalized nanocrystals (“secondary dots”) also have strands of polynucleotides of known sequence extending therefrom, wherein the nucleic acid sequence of the polynucleotide strands of the secondary dots is sufficiently complementary to the nucleic acid sequence of the polynucleotide strands on the primary dots such that, under suitable conditions for promoting contact and hybridization, the respective complementary strands hybridize to each other in forming a dendrimer. In multiple steps in which subsequent additions of functionalized nanocrystals alternate between primary dots and secondary dots, a dendrimer of multiple layers of functionalized nanocrystals is formed, thereby resulting in a detectable signal and an exponential increase in the amount of detectable signal that can be detected from a single molecular probe. Additionally provided, are assay kits comprising reagents for the signal amplification system according to the present invention.
The above and other objects, features, and advantages of the present invention will be apparent in the following Detailed Description of the Invention when read in conjunction with the accompanying drawings in which reference numerals denote the same or similar parts throughout the several illustrated views and embodiments.
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Barbera-Guillem Emillo
Castro Stephanie L.
Nelson M. Bud
Bio-Pixel Ltd.
Miller Raymond A.
Pepper Hamilton LLP
Sisson Bradley L.
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