Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-15
2001-09-04
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S094000, C250S559290, C422S082050
Reexamination Certificate
active
06284465
ABSTRACT:
TECHNICAL FIELD
This invention relates to analytical tools and methods for monitoring levels of gene expression and mutations in gene sequences. In particular, the invention relates to an apparatus, system and method of locating hybridized nucleic acid features on a substrate.
BACKGROUND ART
Immobilized oligonucleotides or polynucleotides of known nucleic acid sequences in an array on a substrate can be used as “probes” for monitoring levels of gene expression or to determine the absence or presence of known or new mutations in gene sequences. Sequences of nucleic acids are synthesized into polynucleotides and oligonucleotides either directly on the substrate (in situ), or indirectly (e.g., pre-synthesized) and deposited onto the substrate into an array pattern using well-known methods. Such methods are referenced below. These oligonucleotides are immobilized on the substrate in the array pattern.
The plurality of probes in each location in the array is known in the art as a “nucleic acid feature” or “feature”. A feature is defined as a locus onto which a large number of probes, all having the same nucleotide sequence are immobilized. The oligonucleotide probes are exposed, for hybridization purposes, to a sample containing nucleic acids of known sequences at unknown concentrations, or unknown sequences, to be tested or evaluated. These nucleic acids are known in the art as “targets”. Note that some investigators also use the reverse definition, referring to the surface-bound oligonucleotides as targets and the solution sample nucleic acids as probes. Henceforth, this application shall use “probes” to describe surface-bound oligonucleotides and “targets” to describe nucleic acids in solution that comprise the analytic sample in some assay or procedure. The nucleic acids or nucleotides in the target sample may be complementary to the nucleic acid or nucleotide sequences in the oligonucleotide probes.
Hybridization is the process where complementary nucleic acids will pair up, associate or bond together. Using well-known processes and conditions for hybridization, the sample nucleic acid “targets”, will hybridize with the nucleic acids of known oligonucleotide probe sequences and thus, information about the target samples can be obtained. The processes and conditions of hybridization between nucleotide sequences are referenced below.
Depending on the make-up of the target sample, hybridization of probe features may or may not occur at all probe feature locations and will occur to varying degrees at the different probe feature locations. After hybridization of the targets with the probe features, the array is analyzed by well-known methods. Hybridized arrays are often interrogated using optical methods. Typically, the targets are labeled using well known methods and with well-known substances, e.g. a fluorophore that will fluoresce when exposed to a light source. The targets are labeled with a fluorophore either before the targets are applied to the array substrate, or labeled with a fluorophore after hybridization with an array substrate, such that the fluorophore will associate only with probe-bound hybridized targets.
Typically, measuring the hybridization to an array of known nucleic acid probes gives valuable information about the target samples. A focused light source (usually a laser) is scanned across the hybridized array causing the hybridized areas to emit an optical signal, such as fluorescence. The fluorophore-specific fluorescence data is collected and measured during the scanning operation, and then an image of the array is reconstructed via appropriate algorithms, software and computer hardware. The expected or intended locations of probe nucleic acid features can then be combined with the fluorescence intensities measured at those locations, to yield the data that is then used to determine gene expression levels or nucleic acid sequence of the target samples. The process of collecting data from expected probe locations is referred to as “feature extraction”. The conventional equipment and methods of feature extraction are limited by their dependence upon the expected or intended location of the probe features on the substrate array, which is subject to the accuracy of the manufacturing equipment.
The scanning equipment typically used for the evaluation of hybridized arrays includes a scanning fluorometer and is commercially available from different sources, such as Molecular Dynamics of Sunnyvale, Calif., General Scanning of Watertown, Mass., Hewlett Packard of Palo Alto, Calif., or 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.
The laser light source generates a collimated beam. The collimated beam sequentially illuminates small surface regions of known location. The resulting fluorescence signals from the surface regions are collected either confocally (employing the same lens used to focus the laser light onto the array) or off-axis (using a separate lens positioned to one side of the lens used to focus the laser onto the array). The collected signals are transmitted through appropriate spectral filters, to an optical detector. A recording device, such as a computer memory, records the detected signals and builds up a raster scan file of intensities as a function of position, or time as it relates to the position. Such intensities, as a function of position, shall henceforth be referred to as “pixels”. The pixels within a region centered upon the expected or intended position of a feature can be averaged to yield the relative quantity of target hybridized to the probe in that feature, if the expected or intended position of the feature is sufficiently close to its true position. For a discussion of the optical scanning equipment, see for example, U.S. Pat. No. 5,760,951 (confocal scanner) and U.S. Pat. No. 5,585,639 (off axis scanner), each incorporated herein by reference.
A general problem in the feature extraction process described above is the extraction of features having weak or low fluorescence intensities, called “dim features”. A feature that yields little or no hybridization to the target sample will produce a low average fluorescence intensity when scanned (i.e. will display poor intensity contrast, relative to a background). However the dim features are just as important in the analysis of genes as are bright features (having extensive hybridization). The majority of genes in a given cell type are expressed at low levels (for example, less than about 50 copies of the gene per cell). Therefore, an array constructed from features that measure expression levels of any plurality of available genes will result in a majority of the hybridized features being dim rather than bright.
If the dim hybridized probe feature is located or positioned accurately in the array and is of known shape, then accurate feature extraction can be performed automatically, using relatively simple algorithms. The computer is programmed to analyze predefined regions of interest on the array based on the expected or intended locations of the probe features that were placed by the manufacturing equipment. The computer will analyze the results of the optical scan by considering the predefined regions of interest. If a pixel within the raster-scan image of a dim feature is within the region of interest, the computer will include the pixel in its data collection.
One problem arises when the probe feature that produces a weak signal after hybridization is not accurately located on the array substrate by the manufacturing process. Although, it is conventional practice to provide fiduciary markings on the array substrate, for example, to which the manufacturing equipment aligns each manufacturing step, errors in the location of the features still occur. The fiduciary markings are also used during feature extraction. T
Agilent Technologie,s Inc.
Horlick Kenneth R.
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