Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-12-18
2002-09-10
Zitomer, Stephanie (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S024320, C435S005000, C435S006120, C435S283100, C435S287100, C435S287200, C435S288700, C422S050000, C422S067000, C422S068100, C422S082050
Reexamination Certificate
active
06448387
ABSTRACT:
Disclosed herein are arrays of polymeric targets useful for evaluating the expression of polynucleotides and polypeptides, and methods of making and using such arrays.
BACKGROUND OF THE INVENTION
Arrays of binding agents, such as nucleic acid molecules, have become an increasingly important tool in the biotechnology industry and related fields. These arrays typically comprise a plurality of binding agents, e.g. DNA or protein, deposited onto a solid substrate surface in a precise array or pattern. Such arrays find use in a variety of applications, including gene discovery, genomic research and bioactive compound screening. One important use of arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in genes expressed in the cells being compared. Such information is useful for the identification of the types of genes expressed in a particular cell or tissue type in a known environment.
The economics of arrays favors a high density design criteria providing microarrays for detection of transcription events for a large number of genes provided that the target molecules are sufficiently separated so that the intensity of the indicia of a binding event associated with highly expressed probe molecules does not overwhelm and mask the indicia of neighboring binding events. Some genes are inherently high expressors, e.g. transcribe mRNA at significantly higher quantities, as compared to other genes which are inherently low expressors. For instance, differences in transcription levels of about an order of magnitude or more are not uncommon. As a result the indicia of binding events on an array from a high expresser intensity probe molecule can obliterate indicia from up to eight neighboring target molecules or more in a rectangular grid.
One object of this invention is to provide arrays in higher density of target molecules without interference from binding events associated with high expresser probe molecules.
Another object of this invention is to provide methods of transcription profiling using high density arrays for full genome profiling without interference from binding events associated with high expressor probe molecules.
These and other objects of the invention will be apparent from the following description and claims.
SUMMARY OF THE INVENTION
This invention provides sets of polynucleotide or polypeptide target molecules immobilized in an array on a surface of a substrate where the target molecules are selected for their capability to hybridize with probe molecules expressed by an organism of interest. A subset of the target molecules is arranged according to intensity at which the organism expresses a set of cognate probe molecules.
In one embodiment the probe molecules comprise a first set of probe molecules which, at a fixed set of conditions, are on average expressed by the organism at a higher concentration than other probe molecules. The first set of target molecules which hybridize to the first set of higher expressed probe molecules is segregated from other target molecules which hybridize to the other probe molecules. In a preferred embodiment the first set of target molecules is segregated at a peripheral region of the substrate. In another preferred embodiment the average expression intensity of the first set of higher expressed probe molecules is at least a factor of 2 greater than the average expression intensity of the other probe molecules. In still another embodiment of the invention the surface density of segregated target molecules on the substrate is less than about 0.5 times the surface density of the other target molecules.
In one embodiment of the invention the arrays are microarrays of single-stranded cDNA target molecules. Such microarrays are especially useful for hybridizing with labeled mRNA probes, e.g. for transcription profiling studies, and can be prepared for a variety of organisms, e.g. animal, plant and/or microorganism.
The invention also provides methods for fabricating arrays of target molecules by segregating to a peripheral region of a substrate target molecules which have been identified by higher than average indicia of hybridization for certain conditions of probe molecule expression by an organism of interest.
This invention also provides a method for detecting a binding event between a naturally expressed probe molecule and a target molecule immobilized in an array on a substrate. The method comprises (a) contacting an array of target molecules immobilized on a substrate with a solution of a plurality of probe molecules expressed by an organism, where the target molecules are arranged on the substrate according to expression level; and (b) detecting binding events between target molecules and probe molecules where the indicia of binding for probe molecules expressed at higher concentration does not interfere with indicia of binding for probe molecules expressed at lower concentration. In preferred embodiments of the method the probe molecules are labeled with radioactive, fluorescent and enzymatic labels.
REFERENCES:
patent: 5143854 (1992-09-01), Pirrung et al.
patent: 5807522 (1998-09-01), Brown et al.
patent: 6040138 (2000-03-01), Lockhart et al.
patent: 6280946 (2001-08-01), Hyldig-Nielsen et al.
Tanaka et al. “Genome-side expressionprofilingof mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray” Proc. Natl. Acad. Sci. USA 2000, 97(16): 9127-9132.
Cambray Jeffrey P
Obar Robert A.
Slater Caroline
Forman B J
Hoerner, Jr. Dennis R.
Kelley Thomas E
Lavin, Jr. Lawrence M.
Monsanto Technology LLC
LandOfFree
Polymeric arrays adapted for high expressing polynucleotides does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polymeric arrays adapted for high expressing polynucleotides, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polymeric arrays adapted for high expressing polynucleotides will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2883293