Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-04-27
2003-02-11
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S005000, C435S007100, C435S007200, C435S006120, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06518056
ABSTRACT:
TECHNICAL FIELD
This invention relates to analytical tools and methods for assaying biological materials in a variety of applications. In particular, the invention relates to an apparatus, systems and a method for assaying biological materials for monitoring levels of gene expression and mutations in gene sequences using an annular format.
BACKGROUND ART
Conventional analysis of biological materials, such as DNA, RNA, proteins and the like, employs an apparatus having biological material on a substrate in an array pattern of discrete features. The features are typically chemically bound to the substrate. The features may be either “probes” of known molecular make-up or “targets” of unknown molecular make-up. For the purposes of simplicity, hereinafter the features bound to the substrate will be referred to as probes and the samples under test will be referred to as “targets”. Arrays of biological probes are quickly becoming a powerful method of simultaneously assaying thousands of targets within a single biological sample. The surface bound probes are typically formed of DNA oligonucleotides, cDNA's, PCR products, antibodies, antigens, and the like.
The arrays are manufactured using automated equipment, such that the spatial location on the substrate of each type of surface bound probe is known within a certain margin of error. The sample containing the unknown quantities of the targets is modified so that each potential target molecule is labeled with a fluorescent label. The sample is applied to the array surface so that the targets may hybridize or bind to their complementary surface bound probe. After the reaction is complete, the surface of the array is washed. The hybridized array is interrogated optically to determine the level of hybridization and the locations and therefore, the identity of the hybridized targets. Optionally, the array substrate may be put into a package for handling, processing and optical interrogation. The array can be held in the package with an appropriate adhesive or glue.
Optical interrogation is typically performed with commercially available optical scanning systems, examples of which are described in U.S. Pat. Nos. 5,837,475, 5,760,951 (confocal scanner) and U.S. Pat. No. 5,585,639 (off axis scanner), all incorporated herein by reference. Typical scanning fluorometers are commercially available from different sources, such as Molecular Dynamics of Sunnyvale, Calif., General Scanning of Watertown, Mass., Hewlett Packard of Palo Alto, Calif. and Hitachi USA of So. San Francisco, Calif. Analysis of the data, (i.e., collection, reconstruction of image, comparison and interpretation of data) is performed with associated computer systems and commercially available software, such as IMAGEQUANT™ by Molecular Dynamics or GENECHIP™ by Affymetrix of Santa Clara, Calif. Typically, a laser beam is scanned across the array surface. The laser beam excites the fluorescent labels on the hybridized targets and the fluorescent signal is detected by a detector and processed by a computer. The intensity of the signal at each physical location in the array is a measure of the hybridization efficiency of a target with a known chemical probe. The intensity relates directly to the concentration of that target within the sample. The signal can be used to simply identify the targets within the unknown or quantitate the targets. The identity of the target is known since it is the complement of the probe.
While there are many methods known in the art for forming and analyzing such arrays, all of them assume that the array will be created and, subsequently analyzed or “read” in “an x, y format”, where “x” and “y” are the coordinate axes of a two dimensional Cartesian coordinate system. “Reading” an array in an x, y format typically requires raster scanning a laser beam across the array surface. Typically, either the laser beam and the fluorescence detection system must be moved or the array must be moved in relation to the optics to scan in a raster pattern or x, y format. In either case, the system typically requires some kind of x, y table or a single axis motion table and a galvanometer.
Typical specifications of a biological array require a very sensitive optical assay. Therefore, the size of the laser beam is normally focussed to about 5 to 10 microns to meet the sensitivity requirements of the assay. In order to scan an array surface in increments of 5 or 10 microns, the x, y table or galvanometer must be very precise. Such precision is normally expensive. The price of an optical scanner is typically above $50,000, which can be cost prohibitive for small analytical laboratories.
Moreover, if an x, y table is employed to move either the optics or the array in a raster scan fashion, the x, y table must be moved in a precise way. In particular, the table must be moved: (i) very quickly in one direction; (ii) it must accelerate up to “reading velocity” prior to the beam touching the array; (iii) then it must move across the array at a constant speed; (iv) decelerate outside of the array area; (v) reverse direction; and then repeat steps (i)-(v) until the surface is completely scanned. A significant portion of the total time required to scan the array is spent in making changes in the direction during the raster scan.
Moreover, in the x, y format, the laser beam must be scanned beyond the region of interest on the array to allow space for the deceleration of the beam and for the beam to reverse direction. Since the array under test is normally glued into a package, the beam may hit a glue edge or line. Conventionally, adhesive glues are often fluorescent and are likely to be more fluorescent than the signals from the very sensitive array. If the laser beam encounters the glue line, the resulting signal can overload the detection system. Overload can result in either temporary degradation of the detection channel or permanent damage.
If a galvanometer is employed, the collection optics must be much larger to collect signal from the entire swept line. Larger optics are more expensive and more likely to have aberrations.
Therefore, it would be advantageous to have an assay system that does not require the complexities and expense of, and avoids the problems associated with, the traditional x, y raster scan format.
U.S. Pat. No. 5,508,200 Tiffany et al., discloses an automated chemical analysis system for high volume chemical testing that creates arrays of chemical reactions on an absorbent media or a solid substrate patterned with microwells. The system includes a dispensing mechanism for dispensing multiple reagents or test samples within a common test area on the media. The common test area has microcuvettes or microwells to hold the samples and reagents and prevent commingling of chemicals between each sample. Mixing of the samples with the reagents is either performed before dispensing or on the media. Tiffany employs a CCD camera to simultaneously monitor the individual reactions at the discrete locations within the common area.
In one embodiment, Tiffany discloses using a rotatable circular disk of an absorbent matrix instead of a continuous strip for the purpose of accommodating multiple sample spots. As a result, photometric measurements with the CCD camera can be made at a single station. The circular disk is rotated to bring the set of microcuvettes into position for photometric measurement. The circular disk format facilitates analyses requiring measurement at variable time intervals and also multipoint rate analyses because of the ease of returning to the single camera station.
However for several reasons, the chemical analysis system described by Tiffany et al. is not conducive to the conventional analysis of biological materials using arrays, as described above. The arrays of biological materials require that each feature be exposed to the entire quantity of the target sample since the concentration of the analytes is typically orders of magnitude less than that of chemistries Tiffany et al. use. The analysis system of Tiffany et al. requires fluid isol
Schembri Carol T.
Schleifer Arthur
Agilent Technologie,s Inc.
Siew Jeffrey
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