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Hybridization-mediated analysis of polymorphismsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidHybridization-mediated analysis of polymorphisms description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128617, Hybridization-mediated analysis of polymorphisms. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional No. 60/470,806, filed May 15, 2003. BACKGROUND [0002] The standard method of genomic analysis for mutations and polymorphisms, including for CF, is the "dot-blot" method. Samples including target strands are spotted onto a nitrocellulose support, and then contacted with labeled probes complementary to the mutations or polymorphic regions. The labels allow detection of probe hybridization to immobilized complementary target sequences, as unbound labeled probes are removed by washing. In another method--a "reverse dot-blot format"--an array of oligonucleotide probes is bound to a solid support, and then contacted with a sample including target sequences of interest. See, e.g., U.S. Pat. No. 5,837,832. [0003] Both methods of assaying mutations or polymorphisms have significant disadvantages. The dot-blot method is itself labor-intensive. It can also yield erroneous results due to the inaccurate reading of assay signals, usually done by autoradiography, which adds further labor, as the probes must be frequently re-labeled. The method described in U.S. Pat. No. 5,837,832 involves a complex and costly on chip synthesis of an array of oligonucleotides, an approach which is better-suited for large-scale genomic analysis and is neither practical nor cost-effective for diagnostic applications requiring only a limited but changing number of probes. [0004] An assay method suitable for multiplexed analysis which avoids many of the problems associated with the above methods involves use of random encoded arrays of microparticles, where the encoding indicates the identity of an oligonucleotide probe molecule bound thereto. See U.S. patent application Ser. No. 10/204,799: "Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays." The bead array is contacted with labeled amplicons, generated from a patient sample, and the labels are then detected (if the labels are fluorescent, the detection can be with optical means) and the bound amplicons are identified by decoding of the array. [0005] In a multiplexed hybridization assay, cross-hybridization among mismatched, but closely homologous, probes and amplicons can generate false positive signals. Thus, the assay should be designed to minimize such effects. A number of mutations and polymorphisms are significant only if they are homozygous, and therefore, to be useful in such cases, the assay must be capable of discriminating heterozygotes from homozygotes. Also, in determining the assay results, where both the encoding method for the beads and the determination of assay results is with optically detectable means, the encoding on the beads can cause spectral leakage, which can be affect the assay signal discrimination. A method of correcting for such spectral leakage is also needed. [0006] Cystic fibrosis ("CF") is one of the most common recessive disorders in Caucasians, with an occurrence of 1 in 2000 live births in the United States. Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are associated with the disease. The number of CFTR mutations is growing continuously and rapidly, and more than 1,000 mutations have been detected to date. See Kulczycki L. L., et al. (2003), Am J Med Genet 116:262-67. Population studies have indicated that the most common CF mutation, a deletion of the 3 nucleotides that encode phenylalanine at position 508 of the CFTR amino acid sequence (designated .DELTA.F508), is associated with approximately 70% of the cases of cystic fibrosis. This mutation results in the failure of an epithelial cell chloride channel to respond to cAMP (Frizzell R. A. et al. (1986) Science 233:558-560; Welsh, M. J. (1986) Science 232:1648-1650.; Li, M. et al. (1988) Nature 331:358-360; Quinton, P. M. (1989) Clin. Chem. 35:726-730). In airway cells, this leads to an imbalance in ion and fluid transport. It is widely believed that this causes abnormal mucus secretion observed in CF patients, and ultimately results in pulmonary infection and epithelial cell damage. A number of mutations are associated with CF, and researchers continue to reveal new mutations associated with the disease. The American College of Medical Genetics ("ACMG") has recommended a panel of 25 of the most common CF-associated mutations in the general population, especially those in Ashkenazi Jewish and African-American populations. A multiplexed hybridization assay for CF-associated mutations in the general population would test for this panel. SUMMARY [0007] Described are practical and cost-effective methods of assay design and assay image correction, useful for multiplexed genetic screening for mutations and polymorphisms, including CF-related mutants and polymorphs, using an array of probe pairs (in one aspect, where one member is complementary to a particular mutant or polymorphic allele and the other member is complementary to a corresponding wild type allele), with probes bound to encoded particles (e.g., beads) wherein the encoding allows identification of the attached probe. The design methods disclosed herein were used to design an assay for CF-related mutations by hybridization-mediated multiplexed analysis, and were extensively validated in many patient samples, and demonstrated to be capable of identifying the most common mutations, including mutations in exons 3, 4, 5, 7, 9, 10, 11, 13, 14b, 16, 18, 19, 20, 21 and introns 8, 12 ,19 of the CFTR gene. [0008] Before hybridization, the region of interest in the genomic sample is amplified with two primers, one for each strand in the region of interest. Of the two strands generated in the PCR amplification step, one is arbitrarily designated herein as "sense" and one as "anti-sense." In certain instances, it is desirable to select, for subsequent mutation analysis by hybridization, either the sense target strand (to be hybridized to sense probes) or the anti-sense target strand--to be hybridized to anti-sense probes. Strand selection is accomplished, for example, by post-PCR digestion of a phosphorylated strand. In particular, strand switching is desirable whenever probe-target combinations (e.g., sense-probe/sense target hybridization) involving a stable mismatch configuration, such as a G-T base pairing, can be avoided. [0009] Also disclosed are methods of selecting probes and amplicons for genetic screening for mutations and polymorphisms. The method of selecting probes and amplicons involves the following steps: [0010] providing a family of single-stranded MP amplicons in which one strand is designated sense and the complementary strand is designated anti-sense, said MP amplicons including amplified segments of the genome on which said genetic mutations or polymorphisms are located; [0011] selecting complementary MP probes for each member of said family of MP amplicons; [0012] examining the degree of homology between either the complementary MP probes or between the family of MP amplicons; [0013] dividing said MP probes into one or more probe sets, and dividing said MP amplicons into sets such that the members of each amplicon set are complementary to the members of one probe set, said division based on avoiding homology greater than an acceptance level between probes in the same set or between MP amplicons in the same set; [0014] performing for each said set of amplicons in turn, the following steps for each MP amplicon in said set, in succession: [0015] (a)(i) determining whether, upon contacting a sense MP amplicon with a probe set which includes a complementary MP probe to said sense amplicon, the degree of cross-hybridization of said sense MP amplicon with other MP probes in the probe set will exceed an acceptance level; and, if not: [0016] (a)(ii) retaining said sense MP amplicon in the amplicon set and the complementary MP probe in the probe set, and repeating step (a) (i) for another MP amplicon in said family; [0017] (b)(i) but if said degree of cross-hybridization does exceed said acceptance level: replacing, in the probe set, the cross-hybridizing MP probe with the complementary anti-sense MP probe, and replacing, in the amplicon set, the complementary sense MP amplicon with the anti-sense MP amplicon complementary to said anti-sense MP probe, and [0018] (b)(ii) repeating step (a)(i) and if the degree of cross-hybridization is within the acceptance level: retaining said anti-sense MP probe and corresponding complementary anti-sense MP amplicon in their respective sets and repeating step (a)(i); [0019] (b)(iii) but if the degree of cross-hybridization exceeds the acceptance level after repeating step (a)(i): determining whether, upon contacting said anti-sense MP amplicon with the MP probes in any other set, the degree of cross-hybridization is within the acceptance level, and if so, placing the anti-sense MP probe complementary to said anti-sense MP amplicon into said set and placing said anti-sense MP amplicon into the set of complementary anti-sense MP amplicons; but if the degree of cross-hybridization exceeds the acceptance level following such determination for each existing probe set, reverting to the original sense MP probe and complementary sense MP amplicon and placing said sense MP probe and said complementary sense MP amplicon each into a new set, and [0020] (c) repeating steps (a) to (c) for another sense MP amplicon in said family. [0021] Also disclosed is a method for design of pairs of probes (with a member respectively complementary to a mutant and a wild type amplicon) for hybridization to labeled amplicons generated by amplification of samples and wild type controls. For each anticipated variant, probes are provided in pairs, with one member complementary to the wild type sequence and the other to the variant sequence, the two sequences often differing by only one nucleotide. One method to enhance the reliability of hybridization-mediated multiplexed analysis of polymorphisms (hMAP) is to determine the ratio of the signals generated by the capture of the target matched and mismatched probes and to set relative ranges of values indicative of normal and heterozygous or homozygous variants. [0022] The method set forth above for selecting probes and amplicons for genetic screening for mutations and polymorphisms, can be included as part of a method to select probe pairs (wild-type and variant), by including the following steps in the afore-described method: [0023] providing a family of single-stranded WT amplicons in which one strand is designated sense and the complementary strand is designated anti-sense, said family representing respective amplified segments of a wild type genome which corresponds to each of the amplified segments of the genome which was amplified when producing the family of MP amplicons; [0024] providing and selecting a sense or anti-sense WT probe so as to have both a sense WT probe and a corresponding sense MP probe in the same probe set or, or an anti-sense WT probe and a corresponding anti-sense MP probe in the same probe set; [0025] determining: (i) whether the degree of cross-hybridization between a MP amplicon and a corresponding WT probe in a probe set, and between a WT amplicon and a corresponding MP probe in a probe set, will exceed the acceptance level and, if so, (ii) determining whether said degree of cross-hybridization will fall within the acceptance level if the selected sense or anti-sense MP and WT probes are replaced with the complementary WT and MP probes; and if so, (iii) determining whether said complementary WT and MP probes will exceed the acceptance level for cross-hybridization with amplicons complementary to other members of the same probe set, and if so, (iv) determining whether placing the complementary WT and MP probes into another probe set will exceed the acceptance level for cross-hybridization with amplicons complementary to other members of the same probe set, and if not: retaining the complementary WT and MP probes in said probe set; but if so, (v) repeating step (iv) for each existing probe set, and if said acceptance level is exceeded for each existing probe set, placing the complementary WT and MP probes into a new set and placing the complementary WT and MP amplicons into a corresponding new set. [0026] Cross-hybridization is a concern in -any assay involving multiplexed hybridization, and methods to avoid its deleterious effects on assay results are included herein. One method to correct for cross-hybridization in an array format, is to set a series of temperature increments, selected such that at each temperature, probe-target complexes containing particular mismatch configurations will denature, while those containing matched ("complementary") base pair configurations will remain intact. The signals generated by captured labeled strands hybridized to probes in the array are then monitored and recorded at each temperature set point. Analysis of the evolution of differential signals as a function of temperature allows correction for each mismatch expected to become unstable above a certain "melting" temperature. After all set points for all mismatches are determined, data gathered at lower temperatures can be corrected for all mismatches. [0027] In another aspect, because the assay method herein relies on encoded beads to identify the probe(s) attached thereto, and the encoding in one embodiment is by way of dye staining, the assay signals are often produced by using fluorescent labels and removing background contributions. Specifically, a method of correcting the assay image is disclosed. That is, within the spectral band selected for the recording of the assay image, the recorded set of optical signatures produced by target capture to bead-displayed probes in the course of the assay are corrected for the effects of "spectral leakage" (a source of spurious contributions to the assay image from the residual transmission) of intensity emitted by bead-encoding dyes of lower wavelength. An assay design is provided herein in which a negative control bead is included in the random encoded array for each type of encoded bead that produces unacceptably large spectral leakage, for example, for beads containing different amounts of specific encoding dyes. [0028] In the examples described herein, negative control beads display an 18-mer polynucleotide in order to serve a secondary purpose, i.e., to permit correction of assay images for the effects of non-specific adsorption. Preferably, the background correction is performed by constructing a background map based on the random locations of each type of negative control bead, where each such type of negative control bead is included in the array at a pre-selected abundance. For each type of negative control bead within the array, a background map is generated by locating the centroids of the beads of that type, constructing the associated Voronoi tessellation by standard methods (as illustrated in FIG. 3; see, e.g., Seul, O'Gorman & Sammon, "Practical Algorithms for Image Analysis," Cambridge University Press, 2000; at page 222; incorporated by reference) and then filling each polygon which includes a bead with the intensity of such bead to produce a map (see, e.g., the map shown in FIG. 3). Optionally, standard filtering operations may be applied to smooth the map; that is, to average out effects from neighboring pixels. See, e.g., Seul, O'Gorman & Sammon, "Practical Algorithms for Image Analysis," Cambridge University Press, 2000 for description of a filter). [0029] Such a map represents a finite sample of the entire background contributions to the assay image in a manner that accounts for certain non-linear optical effects associated with arrays composed of refractive beads, which effects are especially pronounced when the beads are placed into mechanical traps on a substrate surface. In addition, background maps will indicate non-uniformities in the background which may arise, for example, from non-uniformn illumination or non-uniform distribution of target or analyte placed in contact with the bead array. Maps for negative control beads of: different types, i.e., containing different amounts of encoding dyes and producing different degrees of spectral leakage, may be normalized to the same mean intensity and superimposed to increase the sampling rate. [0030] The assay image may be corrected as follows by employing the background map. In certain instances, the map is simply subtracted from the assay image to produce a corrected assay image. In other embodiments, the background can be combined with a "flat fielding" step (See, e.g., Seul, O'Gorman & Sammon, "Practical Algorithms for Image Analysis," Cambridge University Press, 2000). In this procedure, the constant (i.e., the spatially non-varying) portions of the background map and assay image are subtracted, and the corrected assay image is divided by the corrected background map to obtain a "flat fielded" intensity map. BRIEF DESCRIPTION OF THE FIGURES [0031] FIG. 1 shows the results of hybridization of 29 different CFTR mutations; where the smaller open bars represent mutant hybridization, and where hybridization to the "normal" is represented by the larger black bars (e.g., EX-10 has a high degree of mutant hybridization). [0032] FIG. 2 shows the results of hybridization of 29 CFTR mutations, with the mutations being different from those shown in FIG. 1. [0033] FIG. 3 shows a background map of negative control carriers for correcting array images. DETAILED DESCRIPTION Continue reading about Hybridization-mediated analysis of polymorphisms... Full patent description for Hybridization-mediated analysis of polymorphisms Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Hybridization-mediated analysis of polymorphisms patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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