TW200538552A - Hybridization-mediated analysis of polymorphisms - Google Patents

Abstract

Described are 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 methods related to avoiding cross-hybridization by selection of probes and amplicons, as well as separation of reactions of certain probes and amplicons where a homology threshold is exceeded. Methods of correcting a fluorescent image using a background map, where the particles also contain an optical encoding system, are also disclosed.

Description

200538552 说明 、 Explanation of invention: Related applications

This application claims the priority of M Application No. 60/470, 806 filed on May 15, 2003. [Technical field and prior art to which the invention belongs] For the analysis of mutations or polymorphisms (including CF) in the genome, the method is "dot-blot," which includes the right and quasi-methods. Specimens of strands) are spotted on a nitrocellulose support, and the y mutation or polymorphic region is complementary to the calibration probe. This = can be detected and can be detected with the immobilized complementary target The probe for sequence hybridization "the target calibration probe is removed by washing. On the other side, the unbonded species" reverse point-decompression (] reverse dot-Motfroma) In the middle, a probe array is connected to a solid support, and then the specimens of the sequence to be labeled are contacted. See, for example, U.S .: 2: Secondary: 5, 837, 832. Seeking a profit. Both methods for analyzing mutations or polymorphisms have significant disadvantages. The point-and-break method itself is labor-intensive. It also obtained incorrect results due to incorrect test signal test signals. Beans are used in automatic radiography to "know more labor" because probes must often be re-calibrated. The method described in: National Patent No. 5'837,832 involves the synthesis of a nuclear and spinal acid array on a complex and Amber wafer, which is a more poetic large-scale (howling genomic analysis, and it requires only limited The diagnostic application for changing the number of probes is not practical or cost-effective. 200538552 A test method suitable for inul tiplex analysis that avoids many of the problems associated with the above methods uses a random coded array of microparticles, The code indicates the identity of the oligonucleotide probe molecule bound to it. See US Patent Application Serial No. 10/204, 799: Analytical method using multiple analysis molecules using a special random particle array ( Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays). The bead array is contacted with a calibrated repeating subcarriers (amplicons) generated from a patient sample, and then the calibration object (if the The calibration substance is a fluorescent substance, which can be detected optically), and the bonded amplicon (amp 1 icON) can be identified by array decryption. In a heterogeneous heterogeneous analysis (mismatched but nearly homologous cross-hybridization), probes and amplicons can produce false positive signals. Therefore, the analysis needs to be designed to reduce Such effects. Some mutations and polymorphisms are significant only when they are homozygous (h〇m〇zyg〇us) ^ Therefore, in order to be useful in such cases, the analysis method must be able to distinguish the heterotype Zygotes and homozygotes. At the same time, when judging these analysis results, the method of encoding the beads and the analysis result are determined by optically detectable methods. The encoding set on the beads can cause spectral leakage. (Spectral leakage), as the identification of the test signal. Therefore, there must be a method to correct the spectral leakage. Cystic fibrosis ("CF") is the most common hidden disease in the Caucasians. One, it occurs in the United States at a rate of 1 per 2,000 surviving newborns. In the cystic fibrosis transmembrane conductance regulator 200538552 (transmembrane conductance regulator; CFTR) Due to mutations associated with the disease. The increase in the number of CFTR mutations is continuous and rapid 'and to date more than 1,000 mutations have been detected. See Kulczycki LL et al. (2003) in Am J Med Genet 116: 262 -67 published research. Population studies indicate that the most common cystic fibrosis mutation (a deletion of 3 nucleotides at the 508th position encoding phenylalanine in the CFTR amino acid sequence (designated ΔP508)) and Approximately 70% of cystic fibrosis cases are associated. This mutation causes dysregulation of epithelial air ion channels to cAMP (Frizzell RA et al. (1 986) Science 233: 558-560; Welsh, M.J. (1986) Sience 232: 1648-1650; Li, M. et al. (1988) Nature 331: 358-360; Quinton, PM (1989) Clin Chem. 35: 726-730). In tracheal cells, this mutation results in an imbalance between iron ions and body fluid transport. This is widely believed to cause abnormal mucus secretion in patients with cystic fibrosis and ultimately lead to lung infections and epithelial cell damage. Some mutations are associated with cystic fibrosis, and researchers continue to uncover new mutations associated with the disease. The American College of Medical Genetics ("ACMG") has proposed 25 panels of the most common cystic fibrosis-related mutations in the population, especially those of Central European Jews (Ashkenazi Jewish) and non- African-American ethnic group. This control panel will be tested for diversity hybridization analysis of cystic fibrosis-related mutations in general ethnic groups. [Summary of the Invention] Overview 200538552 describes experimental design and experimental images. Methodology of correction! This J Wenyi 'Effective diversity genes for mutation and polymorphism: selection of mutations and polymorphisms' is an array of probe pairs (in-aspects' which: members can With a specific mutation or polymorphism of the dual gene I—the members can be complementary to the corresponding wild-type dual gene), the probe is bound to a microparticle (eg, a bead) encoded by a warp, where the edit can be Alder needles. The design method disclosed here is used to design-test against CF-related mutations (by analyzing the diversity of hybridization intermediaries) 'and in many diseases Extensive and effective mutations in human specimens that have been shown to recognize the mutations included in exons (CF) of the CFTR gene 3, 4, 5, 3, 9, 10, 11, 13, 14b, 16, 18, 19, 20, 21 and introns 8, 12, 19 Before the hybridization reaction, two primers are used to amplify the desired region in the genome sample. Each primer is paired with the desired gene. In the step, The resulting two strands are arbitrarily designated here as "sense" and the other as "anti-sense." In some instances, mutations in order to later use hybridization reactions For analysis, it is necessary to select the stock of the meaning stock (hybridized with the sense stock probe) or the stock of the antisense stock—the heterosexual parent of the antisense stock probe. The selection of the stock can be used, for example, after pcR digestion (such as PCR digestion) to achieve phosphorylated strands. Especially when probe-target binding (such as the hybridization reaction of meaning stock- 楝 needle / significance stock) involving a stable mismatched configuration (eg GT base pairing) can be avoided At the same time, strand switching is satisfactory. The present invention discloses a method for selecting a probe for genetic and genetic mutation screening and 200538552 amplicon selection. The method for selecting a probe and amplicon involves the following provision-single-strand MP amplicon family, one of which is The complementary strand is selected as an antisense strand, and the MP amplicon contains an amplified genomic fragment on which there is a mutation or polymorphism of the gene; it is selected for each member of the MP amplicon family Complementary MP probes; test for homology between complementary MP probes or between Mp amplicon families

The MP probe is divided into one or more probe groups, and the MP amplicons are grouped so that members of each group of amplicons and members of the probe group; complement, the grouping method is based on avoidance The homology is greater than the acceptable degree between the probes of the same group or between the MP amplicons of the room group; the human amplicons are used as the amplicons of each group, and each amplicon in each group is combined with the next step : (A) (i) Amplification based on a sense strand Mp

The contact of the complementary probe sets determines the MP amplification of the sense strand. ^ Is the degree of cross-hybridization of other MP probes in the bismuth needle more than? The degree of crossover, if not: Two points (8) (11) The significance MP amplicon retained in the amplicon group and the complementary Mp probe in the 针 k-needle group, and the family of amplicons Repeat step (a) ⑴; 卯 Extend • 1) But if the degree of cross-hybridization is higher than the acceptable level _ 罙 Needle group, replace the MP of the cross-parent with the complementary antisense MP probe 4 丄_,, Ten, and in the amplicon set, the antisense strand MP amplicon complementary to the antisense strand MP probe 11 200538552 replaces the complementary sense strand Mp amplicon, and (b) ( ii) Repeat steps (a) and (i), assuming that the degree of cross-hybridization is within reachability. The antisense stock good probes and relative complementary antisense stocks retained in their respective groups MP amplicons, and repeating the steps; (b) (iii) but assuming that after repeating steps (&) (1), the degree of cross-hybridization exceeds the acceptable level: according to the antisense MP amplicons and The contact of the MP probe in any group determines whether the degree of cross-hybridization is acceptable, and if so, the complement of the antisense strand Mp amplicon is complementary. The sense strand MP needle is placed in this group, and the antisense strand Mp amplicon ^ is set to the 4 complementary antisense strand Mp amplicon group; but it is assumed that according to the determination method for each existing probe set Beyond the acceptable level of cross-hybridization, repeat the original sense stock MP probe and the complementary sense stock Mp amplicon and place the Sisi stock MP probe and the complementary sense stock Mp amplicon into a new group And (C) in this family, repeat steps (a) to (C) for amplicons of other meaningful strands. At the same time, the present invention discloses a method for designing a probe pair (there is a Or wild-type amplicon complementary members), this probe is used to hybridize with the labeled amplicon (produced by the amplification reaction of the sample) and the wild-type control group. For each predictable change, the probes are provided in pairs, which can be complementary to the wild-type sequence '(the other is complementary to the variable sequence), and the two sequences usually differ by only one nucleotide. A method to enhance the credibility of the polymorphic hybrid intermediary diversity analysis (hMAP) is the proportion of m number (12 200538552 of this signal is generated because of the capture of the target pair and the mismatched probe), and the setting can be shown on the page The range of values associated with positive $ and heterozygous or homozygous variants. The method of selecting probes and amplicons for gene screening using polymorphism and polymorphism can be described as part of the method for selecting probe pairs (wild type and variant type). 'Add the following method to the aforementioned method:

Provide—A family of single-strand wt amplicons, in which one strand is selected as a sense strand and the complementary strand is warped as an antisense strand. Each of these families expresses a male f-type gene, an amplified portion of the group & For each part of the genome that is amplified, it is amplified when a family of MP amplicons is produced; provision and selection of a sense strand or an antisense strand WT probe to have a sense strand WT probe at the same time Needle and a relative sense unit such as: ― 'or in the same probe set at the same time have an antisense stock subscription probe person an opposite antisense stock MP probe; Fu Xi: (i) a probe π, Zintandan I; the degree of cross-hybridization between two needles' and one dozen in a probe set

The degree of cross-hybridization between the two-phase material MP probes, whether it is in range, and if so ((assuming that the selected meaning stock or anti-moon up and WT probes are replaced with complementary _ Mp_ > mu two-way: Whether it will fall within an acceptable range; (ιιι) Determines whether the complementarity, whether the probe and an extended two-hybrid that can be complementary to other members in the same probe set will exceed the acceptable level, and it is assumed to be yes. Mutual WT and MP probes are placed in another probe set, and the degree of cross hybridization between two complementary amplicons of two other shellfish complementary amplicons will be reached again, and if not, the complementary n and MP Probe 13 200538552 In this probe set; make coffee < — fake government as a sham, (V) repeat step A (1 V) for each existing probe set, and falsely order Syria. If the probe set existing on the parent exceeds the acceptance level, the complementary WT and MP control # # s ^ i

, Put ten into a new group, and put the complementary Π and MP amplicon into a corresponding new group. Miscellaneous is also related to methods that involve multiple hybridizations in any analysis and that avoid adverse effects in the analysis results. A method of cross-hybridization in an array format is to set a series of temperature increasing conditions, and select the one that contains a specific mismatched configuration at 1 degree per field.

The temperature at which the probe-target complex will denature, and those that are paired ("Complementary test base pairs will maintain 者 #. Order a text ..., and then detect the heterogeneous parent to capture the calibration of the probe on the array ^ ^ The horror of the house produced, and at the temperature set by the mother, the evolution of the different signals of the "knife analysis" as a function of temperature can be corrected beyond a certain melting point / dish. Unstable every wrong pairing. When all the warm sounds of all wrong pairings have been determined, the data collected from a lower temperature can be corrected for all wrong pairings .

In other respects, because the analytical method here relies on coded beads to identify the probes attached to it, and the code is-in a specific example, stained with a dye, the test signal is usually fluorescent Signal calibration and removal of background values. In particular, the present invention discloses a method for correcting and analyzing images. That is, in the spectral band selected to record the test image, the "spectral leakage amount (the amount of spectral leakage ( spectrai leakage) "(the source of a few distorted contributions from the residual emission of the analysis image), the intensity is released by the low-wavelength bead code 14 200538552 agent. An experimental design is provided here. The negative control set of beads is included in a random coded array of each form of coded beads. The coded beads produce an unacceptably large amount of spectral leakage (for example) containing different A number of beads specifically coded for the dye. In the example described herein, the negative control group beads exhibit an 18-mer polynucleotide for a second use, that is, to allow correction of experimental images of non-specific absorption effects. Preferably, the background value correction is to construct a background image image according to the random position of each type of the negative control group beads, wherein each of the types of the negative control group beads is pre- Selective abundance is included in the array. For the mother type of the negative control group beads in the array, a background image image is passed through the center of mass of the bead located in this type using a standard method (as shown in the third figure; see, for example, Seul, O'Gorman & Sammon, "Practical Algorithms for Image Analysis," "Cambridge University Press, 2000; p. 222; incorporated as reference material" constructs the relevant Voronoi checkerboard pattern and then fills it Each polygon containing a bead, the bead having the intensity of the polygon can produce an image image (see, for example, the image shown in the third figure). Optionally, standard transition operations can be applied to smooth the image; that is, to average the effects from neighboring image values. (See, for example, Seul, 0, Gorman & Saminon, "Practical Algorithms for Image Analysis, 55 Cambridge University Press, 20000 for a description of filters." A limited sample is presented in all background values to construct 15 200538552 The analysis image method, / ^. T. ~ The method calculates some non-linear visual effects, which are related to the resulting array. The bead was placed and the surface of the mechanical catcher was specifically declared. In addition, the background value image will show the non-uniform burdock that may rise in value in April, and since Non-uniform brightness or non-uniform target distribution or placement, analysis of human] bead array contact (analy). Used for different types of negative control group images, that is, the encoding Agents and produce different degrees of spectral leakage, " if w ^ the month b is normalized far more than the same average intensity and is added at a rate that increases the specimen. The analysis image may be corrected by successively using the background image In In the embodiment, the image is simply removed from the analysis image to generate a corrected analysis image. In some specific examples, the background value may be, flat field method, (See, eg, Seul, O'Gorman δ Sammon, "Practice Algorithms for Image Analysis," Cambridge University Press, 2000). In this process, the constant (that is, the spatial non-variability) becomes a part of the background value image, the test image is removed, and the corrected analysis image is segmented via the corrected background image to obtain A "planar field method, intensity image. Detailed description of the invention Provided herein is a multiplexing of a designed set polymorphism (hMAp) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Analyze intermediary hybridization methods. Probes used to detect mutations in a target sequence, and repeater probes with high relatives 16 200538552 power, when the entire repeater probe or one of its repeaters (subseed u: e) When it is completely complementary ("paired") with the probe, it is shot to the selected target position (designated with state We); As a result: when it hybridizes with-low affinity repeater probe, it has incomplete = "Mistaken pairing") region. In general, the probe of the present invention must be long enough to prevent sticking to unrelated #MA target sequences. In some specific embodiments, the length of the probe can be about 10 纟 50 Test basis, or preferably about Η to 25 bases, and more preferably 18 to 20 bases.

Probes use linker moieties to encode microparticles (beads) coded by the glutamate on the 5 'end of the linker moieties in a known manner. The microparticles encode a chemically or physically The resolving properties uniquely identify the attached probe. The probe system is designed to capture a desired target sequence 'which is contained in a solution in contact with the beads. Hybridize to: The target of the probe displayed on a particular bead will produce an optically detectable signal. The optical signal of each participating bead uniquely corresponds to the probe displayed on the bead. Before or after the hybridization step, the operator can determine the identity of the probe using particle identification and detection methods (for example, using decoding or multi-color fluorescence microscopy). The composition of the beads includes (but is not limited to) plastic, ceramic, glass, polystyrene, fluorenyl styrene, acrylic polymer, paramagnetic material, thoria sol, carbon graphite, titanium dioxide, latex Or cross-linked glycan (such as sepharose), cellulose, nylon, cross-linked colloidal particles, and Teflon. See by Bangs Ub〇rat () ries,

Fisher IN's "Microsphere Detection Guide." The particles do not need to be spherical and can be Xuanru] _ to milliseconds (= the size of sex can be in the range of nanometers (eg) (] such as · lmm), compared to Preferably, the beads are from R ^ spoon to about 2GG microns, and more preferably from about Q.5 microns to about 5 microns. In some specific examples, the beads may be Arranged on the soil before the hybridization step into a planar array. Beads can also be loaded on a planar substrate for imaging after the hybridization step. The process and system here provides a high-powered analysis method that allows all The array beads are immediately visualized, and the genetic analysis of the patient's specimen is performed at the same time. The bead array can be a random coded coding array = code (placed in a position in the array that is not known in advance = the beads). The identity of the probe for collecting nuclear and acid. The random coded array can be entered according to the methods and procedures disclosed in International Application No. PCT No./201079 (herein incorporated by reference). Bead arrays can be manufactured using discrete batch processing ( Qing ilcatlón-speciflc) substrate (for example: wafer-specific wafers), and beads with a chemical code and attached to an oligonucleotide probe (eg, about 1 in each specification &suspension; 1) 8 beads). These beads are bound to the substrate (eg, silicon wafers) and form a uniformly implanted array in a selected matrix region. In some specific examples, the The size of the bead array of beads is 300 " by 300 " m. The density will change with the use of beads of different sizes. Multiple bead arrays can also be formed in separate liquid zones at the same time. This method is based on U.S. Patent No. 2 ig / i 92,352 discloses that the name is "microparticle array and its method" 18 200538552, which is incorporated herein by reference. Bead arrays can be formed by the method "leapstm", such as the United States Patent Nos. 6,251,691, 6,514,771, 6, 468, 81 are incorporated by reference. The substrate (eg, wafer) used in the present invention may be a mode of a planar electrode ( patterned), modularization of the interface with LEAPS (interfacial patternin g) The method is consistent. It is a patterned growth (for example) of oxide or other dielectric substances (patterned gr () wth), which produces the desired resistance gradient structure under the supply of AC electric field. The patterns can be Designed to produce the desired aC domain-induced flow configuration and associated particle transport. The substrate can be modularized to wafer scale using semiconductor process technology. In addition, substrates can be placed using one—scribing, Divided by a light-transmissive polymer film, fixed on the substrate with a desired liquid conduit and block layout, so that the liquid is confined to one or several separate blocks, so it can be accommodated on a provided substrate Multiple specimens. 77 罘 一 Planar electrode, ^

The bead array is available to provide

The electrode system is substantially parallel to a second electrode ("sandwich,"), has two electric electrodes separated by a gap, and contains a polarizable liquid culture medium such as an aqueous electrolyte solution. The surface or internal name of the two planar electrodes can be changed by interfaeial patterning. The beads are introduced into the gap. When an alternating current is applied to the gap, the beads are in the first place. Two electrodes (such as

(, Such as · Japanese-Japanese film) to form a random weaving I chemical array. Moreover, LEAPS 'can also be used to form a bead array on a light-sensitive electrode ("wafer". Preferably, the above-mentioned sandwich configuration also uses a planar light-sensitive electrode and other planar electrodes. Once again, this February 19 200538552 The poles are separated by a gap and contain an aqueous electrolyte solution. A functionalized and coded bead is introduced into the gap. Using AC voltage and light, the beads form an array on the light-sensitive electrode In some embodiments, the beads can have a chemically optically distinguishable feature. For example, they can be achieved by staining the beads with some optically resolvable labels, such as those containing one or a The above photoluminescence can distinguish fluorescent or chromophore dyes whose excitation wavelength, emission wavelength, excited state lifetime or radiation intensity. The optically distinguishable label can be used to dye beads in a specific ratio, for example , As described in US Patent No. 4, 71 7, 655 (January 5, 1988) by Fulwyler. Dyeing can also be achieved by increasing the particle size using methods familiar to the skilled person, (Molday, Dreyer, Rembaum & Yen, J. Mol Bio 64, 75-88 (1975), L. Bangs, 'uniform iatex particles, Sergen Diagnostics, 1 984). For example, Up to 12 forms of beads are coded using two color swell and bulk stains, each within four intensity gradings and mixed with four nominal mole ratios Alternatively, the method of encoding the combined color (detailed in International Application PCT / US No. 98/1 10719, incorporated by reference) can be used to give the bead array an optically distinguishable label. In addition to chemical encoding, beads can also be made magnetic by the method detailed in International Application No. WO / 01/098765. In addition to the chemical encoding of dyes, beads with certain nucleotide-priming primers can also be used. Separated by space ("spatial encoding"), so that the position of the beads provides some information about the identity of the beads placed in it. 20 200538552 For example, 'Spatial encoding has In the middle of a process System ,, which by using ί}? Αρς * 4 Shang, late night with LEAPS for phase variation of electric field according to the light projected on the substrate in poor grip + t and / or the structure). ^ μ formula to assemble a flat array (any desired impedance at the interface between the LEAPS chip and the liquid creates a lateral gradient 'to ease the force of the electromechanical mechanics of the intermediary array combination. The requirement of t is moderate: in the liquid gap (Usually 10Mm between two planar electrodes) Apply a low AC voltage of typically low ⑥10Vpp. This combination process is fast and optically programmable • gram containing thousands of beads Arrays can be formed in the electrical domain in seconds. The formation of SUbarrayS can also occur in multiple liquid phases maintained on the surface of the divided wafer. After the array is formed, the array can be immobilized. Examples For example, a bead array (for example, M) can be applied with a direct current to generate a random-encoded array to immobilize it. The DC voltage is usually set to 5 —η (for beads in the range of 2 — 6 #m, and the gap is Those with a size of 100-150 # m), and the supply time of less than 30 seconds in the "reverse bias" structure, so that n-doped Shibajibei can form a positive electrode, resulting in the array Compressed to fit The extent to which the contact between adjacent beads is promoted in the column, and at the same time causes the beads to move to the high electric field area, close to the electrode surface. Once in a sufficiently close situation, the beads are used to guide the physical adsorption of van der Waals force. This adsorption process can be facilitated by providing a group of "tethers" on the bead surface that extend from the bead surface; polylysine and streptavidin have been used for this purpose. 21 200538552 In some specific examples, the particle array can be immobilized by chemical methods, for example, by forming a composite gel-particle film. In an exemplary method for forming a shihaimin-composite particle film Kappa is a microparticle suspension, which also contains all components used for subsequent in-situ gel formation, ie monomers, cross-linking agents and initiators. The particles are assembled using the LEAPS method in the assembly A plane on a substrate; for example, a voltage in the range of 100 Hz to several thousand Hz is provided between the electrodes of the liquid gap in the range of 1-20Vp_pAC voltage. After the array is combined, the cells are heated thermally in the presence of AC voltage ~ 40-50 ° C (Using a far-infrared lamp or a mercury lamp source) to induce a liquid phase polymerization reaction, so that the microparticle array is effectively trapped in the gel. The gel can be made of various monomers such as aery 1 amide and bisacryiamide. Mixtures with a body concentration from 20% to 5% (acrylamide: bisacrylamine = 37.5: 1 'mole ratio), or any other low viscosity water-soluble monomer or monomer mixture can also be used The chemically immobilized and functionalized microparticle arrays prepared through this process can be used in a variety of biological assays, such as ligand receptor binding assays. In a consistent embodiment, a thermal hydrogel is formed by using az〇diis〇butyramidine dihyfrochlorise as a thermal initiator at a low temperature, so as to ensure that the entire ionic strength of the polymerization reaction mixture falls within ~ (In the range of MmM to 1.0 mM. The initiator used for the UV polymerization is Irgacure 2959® (2-Hydroxy-4, -hydroxythoxy-2-methylpropiaphenone, ciba Geigy, Tarrytown, NY). The starting agent is added to the monomer to obtain a solution weight of 5%. In some specific examples, the microparticle array can be fixed by mechanical methods. 22 200538552 For example, a microwell array can be calibrated by standard The semiconductor process method is produced in the low-impedance region of the silicon substrate. Particle arrays can be formed using these structures, for example, by using LEAPS to mediate hydrodynamics and pondermotive forces to transport and accumulate particles on the small hole array. Close the Shaw AC domain 'particles fall into the microwells, thus forming a mechanical constraint. The excessive beads are removed, leaving a geometrically-organized random bead array on the surface of the substrate. A matrix (eg, a wafer) can be placed in one or more closed sections to allow internal contact. Interactions can also be performed in a microtiter plate similar to a microtiter plate. The reaction chamber can be opened in the open section. The reaction reagent can be dripped on the surface of the wafer using an automatic liquid processor, and multiple samples can be processed at the same time. This form provides standard sample processing procedures for the existing microtiter plate format and Liquid processing, combining sample processing and array detection. Coded beads can also be combined on the surface of a substrate (but not inside the array). For example, by using bead suspensions in multiple areas of the substrate Inside, and allow beads to sediment by gravity, bead combinations can be formed on the base_negative. Compared to using bead arrays to form bead arrays, these combinations are generally considered as chaotic structures with low density or non-planar structures, involving stacked or Blocked beads thus prevent imaging of the affected beads. However, the combination of space and color coding (using a mixture of chemically coded beads) Even heavy separation areas on the surface of the substrate) still allow for diversity. In some specific examples, decoded array image comparisons can be used to show chemically or physically distinguishing features, and probe extensions. For example, 23 200538552 That is, such a comparison can be made through the optical microscope of the analysis instrument; into: two = computerized image capture of the extended optical signal of the Ashi probe; car picking = analysis image is used to determine the chemical and / or Physically separable = the decoded image is used to identify the unique features of the probes displayed on the surface of the beads, which are prepared on the array-this way is not discernible. The identity of the probe on the test particle can be identified, and the algorithm of image analysis can be used in force analysis.

Ψ m ΛΑ ^ f

数据, data. These algorithms can be used; ^ / 矣 J ^ is used to obtain each data in an array. The analysis software will automatically use the bright-zone ~ image of the array as a mold to extract the center of the beads, group the beads according to the type, give a fixed amount of strength to the beads, remove "blemishes," (such as in serum samples The "fractal matrix" of the irregular shape in China, the material produced by the knife analysis, the intensity statistics, and the positive average intensity and related variability of all bead types are evaluated. An embodiment of the algorithm, Filed in International Application No. WO 01/098765.

Alas. Probe hybridization can be shown by, for example, optical changes in the beads bound to the probe. This can be done using calibration methods known in the art, including direct and indirect calibration. In some specific examples, the fluorescent s and dyes can be attached to the nucleoside acid added during the probe hybridization process, which changes the optical signal of the bead to the target probe, such as fluorescent The light intensity changes, thus changing the optical signal of the beads). Types Described herein are methods and compositions for performing accurate multi-analysis on highly polymorphic target areas. Design and Composition of Diversified PCR Reactions 24 200538552 At the position of high polymorphism (1〇ci) Ning Xi 4+ expected to lose # Xi 1 sex & domain retention caused the body closest to the exploratory region of the selected polymorphic region ... wooden needle (when (Adhered to the selected polytype y subsequence) will be adjacent to the polymorphic region and the nucleogenic probe will be incorporated with other adjacent polytype regions.The probe is designed to Polymorphic area two: (interrogate) the combination of the target to the correct target ... ± /, with sufficient length to ensure the specificity and thermal stability of the j when playing. This polymorphic region includes non-dietically-selected regions. The UK selects positions and non-target sequences. This article describes the design of the coverage probe set, which is related to the analysis of polymorphism in the polymorphism in the multiple dissimilarity 4 polymorphism score, as it is for CF carriers. Screened mutation analysis. Here, the coverage group for mutation: diversity includes multiple subgroups (muHipie_ 仏 'maternal subgroup can be combined with a selected region. In the second embodiment

The coverage group contains subgroups constructed to reduce the number of probes in the group, and is also described in detail. Bead-bound probe arrays can be used for a herd of mutant hybridizations — intermediary = analysis' fe Wai is a non-regulated canine k and polymorphism in the cystic fiber transmembrane conductance regulator (CFTR) gene. Each selected mutation in this group is associated with the disease and must be scored independently. In the case of point mutations, a coded needle is provided to determine binding to the selected position, one probe is 'probiotic' and the other probe is bound to the sequence of the mutation or polymorphism target. In some specific examples, as long as the sequence of the sequence 25 200538552 ::::: provided by the coverage probe set is sufficiently approximated, it is not of concern that the configuration of the non-selected region meets the special cake It's up — one by one. In this type of example, all sequence pairs are assigned the same code by the knife. In a preferred embodiment, the probe is bound to the same solid support ("probe library,"). Probes The library can reduce the number of discriminable solid supports, which are used to make a statement. In-particularly better specific real estate, the provided solid holders are in groups Or in the form of a distinguishable pico instance array, which can be decoded ::: in situ. The additional polymorphism in the area marked by the "" added in the coverage group: It is considered to indicate that the multiple ethnic groups are single. A useful method for haplotypes. Appropriate probes can be designed to correspond to known dual genes in the CFTR gene position. Some polymorphisms and mutant dual genes are known and can be obtained from the literature and other sources. Standard methods of temperature control can be easily applied to set operations (or apply a series of pre-set temperature changes to) μ degrees of a single wafer or multiple wafer carriers. When combined with direct images of the entire array of coded beads (as provided in the READTM format in the diversity analysis), the application of a preset temperature cycle provides a magnification of the reabtime on-chip extended product in time. Given the genome, mitochondria, or other DNA, the amplification reaction on the linear wafer eliminates the need for a pre-analytical DNA amplification reaction (such as pcR) and therefore significantly reduces the time required to complete the entire assay. Time-sensitive applications (such as the determination of cadaver heads) can therefore be implemented. More importantly, this method will eliminate the complexity of PCR diversity, with limited steps in many genomic screening and polymorphism analysis. In a preferred embodiment, a liquid cartridge provides injectable specimens and reaction reagents, and temperature control. The designs, compositions, and methods described herein are also related to amplifying the diversity of nucleic acid specimens. In a preferred embodiment, the overlay set of pcR primers (consisting of the initiation and adhesion sequences) is used for the target amplification reaction. Described below is a series of suitable array steps for screening probes and targets for hybridization analysis. Assignment: ❿ Identify (select #)-group of probes, p, to perform one or more "multiplexed" at the same time, allowing the interrogation reaction in nucleic acid sequence hybridization to determine the selected in each group Polymorphic position s = {Sl5 Y, SN; [composition, this position is located at M < or 4 nucleic acid strand T: = {T〗, Y, TM} ("subject ,,"). A collection of targets T, {Ti = (mi, σί); K or = i < or = Μ} is produced by polymerase chain reaction (PCR), which uses design to place multiple polytypes Or mutated to a single pcR primer, provided that the target length li does not exceed the maximum length lmax, and the i-th target Ti with length li further has the following characteristics: a diversity mi, which gives the selected polymorphism (Or mutations), where Σ (i = l; i = M)); and an orientation value σ 丨, where σ of the meaning unit ("ci s"), + ι; or 27 200538552 antisense Sigma ("trans") of σ ^ -1. Primers-Preferably, the mutation analysis involves the interrogation of each selected mutation position s by hybridizing the corresponding target to at least two selected challenge probes P / and V, where at least- The _probe ”has a sequence that can be complementary to a normal (" wild type ") composition, and wherein up to a second probe P / has a sequence that can be complementary to a variant (" mutant, ") composition. When interrogated subsequence regions (except in selected regions) are polymorphic or mutated, ideally a "degenerate" is provided, and the probes are Expected composition pairing. The comparative PA%) can be understood as referring to all probes targeting the k-th 敎 position, so that pk is characterized by having some probes, each of which has an orientation value ^ , In contrast to the target of the same origin. In particular, the probe system is selected, and the probe-target reaction system is loaded: in a manner that can be a strain or one or more reaction groups, each of these reactions: Each system is operated in a -separated container. This method can reduce the reaction of any subtypes that contain, or the position of the mutation or the target of the mutation \ with the selected probe pk corresponding to except Φ. 隹: 、,; does not necessarily produce an optimal configuration (conf iguration) The following test and error analysis method is provided. The strategy provides a basis for systematic optimization of experimental optimization. It includes one of the similarities between the two sequences. Max, again & The key * number, which is expressed as the homology score, and an "interaction is also the maximum acceptable degree of the father, which shows its size in the" outer diagonal (〇ff — diag〇nal) (Magnitude),-Co-affinity PJ / 28 200538552 See U.S. Application No. 10 / 2〇99, Bi Wai Shi Lu uses the name ☆ p for application-specific molecular analysis with multiplex analysis ") shown in the given Ethnic group or the process that has a mutual influence between wooden needles and all targets ::: Any target given and targeted at its heart: Fathers and fathers, assign targets and their corresponding probes to ... There are independent "multipliers (mul-the number can be chosen as much as possible from the" A "hybrid reaction), so ...: small, so that the similarity between the targets in the same container, the column similarity ("Maxifflal homology score") its preset maximum acceptable level ❶ 小 m is small-㈣ capacity ^, cross-hybridize between any given target and needle amount- By shifting the bearing value of this unit and its corresponding probe, allow any other Target reassignment e 1. Some targets may have -v L ΛΛ Γ- ^ There are more than one kind of region, and a target in the array u & 疋 ^ needles can hybridize with it. In such cases, in order to reduce the cross hybridization and competitive hybridization in the same container, by redesigning the primers and reducing such "of fending," the diversity of the target to generate a ( (Or above) smaller targets to replace the original single-rod, each: the new target has a lower diversity of hybridization regions than the original. The following pseudocode of Yuancheng provides a configuration for the reaction The description of the process of trial and error in order to reduce cross-hybridization. * 29 200538552 I-assignment target-and cognate probe-to group c ("container,")

c = 0; DO REFSEQ = selection target 选择 Reduced selection (Γ, 1) S = L (c); Draft directory (L (g), REFSEQ); Randomly select a target sequence from the provided set, T * / / * Remove selected targets from the set * / / Place the selected targets in a new group ("Clan ,, ..." ... 'Execute in a directory format, S * / arrange the targets (REFSEQ, r, HScores ); / * Arrange the remaining targets to REFSEq, use paired permutations or multiple sequence permutation pairs; regression homology, source score * /

Classification target (Hscores, 7) / * Arrange the target sequence according to the increase in REFSEQ related to the homology score; first put the least similar, and finally put the most similar to REFSEQ * / ^ 一 ^ (HHCCRERE, / * Remove the targets from the set according to the increased homology score (,,); to maxHSC〇re), and put them into the table, starting from the top; return the number of targets arranged in the directory, S * / t ) / * Remove the selected subject * /} from the set when (/ is not blank);

If necessary, one or more directories can be deleted, if it contains more than an acceptable number of targets (for example, assuming it is: based on having too many targets in a directory, maxHscore should be reduced ), Remove the target from the lower end of the category or more than the category, and place the target in the collection 7. I. Improving the ethnic group configuration When (i = 0; I <c; i ++) sequentially test the current directory of each ethnic group * / ‘{S-L (i);

Po 仏 Select probes (P, S) 30 200538552 When (S is not blank) T- PopTarget (S); Perform the probe target reaction (Pfs, T) When (each probe P, in PofS) / * Each probe is designed to be paired with at least one target, which means that the probe is homologous to the target; note: refer to the diagonal elements in the co-affinity array * / / * Contact T with all selected probes, preferably arranged in the probe array * / 1 = determines the strength of the interaction (P, T); / * Reduce the large outer diagonal elements in the co-affinity array * / If ((P is not homologous to T) and (I > maxi) / * orientation value of flip probe * / flip orientation (P); flip orientation (TcP); / * flip the orientation of the target homologous to P The value of * / turned PopTarget⑻; / * confirm that the "turned, * * T" in the directory s performs the probe-targeted reaction (p0fS, turned / * makes τ better than all selected probes) Is in the probe array * / compared to each probe P, in PofS) k determines the strength of the interaction (P, flipped T); / * narrows down in the co-affinity array which cannot be as large as the outer diagonal Element * / An up and upside down (Different sources) and (I &max;) directory * / push target (inverted τ, TempList); / * place flip target to temporary 31 200538552 S = TempList; when (S is not blank) {T = PopTarget (S) When (j = 0; j <c; j ++) {正 ①⑼ 吐 ⑼) {The directory S temporarily retains the inverted target ~ / * the target of the soul in the temporary directory * / T) (T)); (PcT ); Directory); Recognize T confrontation in all existing directories except those in the original directory of T, s * / τ a, l = lg); select probes in directory (L); / * select probes in group l Needle ”into the needle's response_L, / * contact T with all selected probes, f (each-probe P, the hidden towel ^ is arranged in the probe array ~ 1 == decides the interaction Intensity of action (P, T); / Reduction of large outer diagonal elements is not acceptable in co-affinity arrays * / If ((P and T are different sources) and (I > maxI)) flip orientation / ** / flip target The bearing value makes the response return to the original state. Flip bearing / * The bearing value of the flip probe is the same as the target T * / Push the target (T, new / * start new ethnic group * /

Inverted orientation (P); / * Inverted probe orientation * / Inverted orientation (TcP); / * Inverted orientation value with the same origin as P * / 32 200538552 [Embodiment] Example I: CFTR test from several patients The extracted genomic DNA is amplified by corresponding primers in a multiplex PCR (mPCR) reaction. The PCR conditions and reaction reagents are as follows: Primer design: one of the primers (meaning or antisense, depending on design considerations, discussed later) is labeled with a label (such as Cy3, Cy5, and Cy5 · 5) at the 5 'end Modifications and corresponding primers for complementary sequences have a phosphate group added to the 5 'end, so that the amplicon can be cleaved by the endonuclease in the target post-PCR process (see below). Hybridization was detected using a dye (Cy3, Cy5 or Cy5. 5) detected in the hybridization product. Multiplex PCR (mPCR) was performed in two groups with the following primers (Table I and Table 11), and the following reaction reagents and conditions were used. The number of exons at the position of the mutation is shown in the left column of Table I and Table 11.

Table I

Artificial Sequence Artificial Primer mPCR Group I Primer: ("Cy" represents the dye tag, and "P" represents the phosphate modification at the 5 'end of the primer) SEQ ID NO.:l SEQ ID NO.:2 SEQ ID NO.:3 SEQ ID NO .: 4 SEQ ID NO .: 5 SEQ ID NO .: 6 SEQ ID NO .: 7 SEQ ID NO .: 8 SEQ ID NO .: 9 SEQ ID NO .: 10 SEQ ID NO: 11

EX-5-l-Cy GTC AAG CCG TGT TCT A GAT EX-5-2-P GTT GTA TAA TTT ATA ACA ATA GT

EX-7-1 -P AC TTC AAT AGC TCA GCC TTC EX-7-2-Cy TAT GGT ACA TTA CCT GTA TTT TG EX-9-1 -P TGG TGA CAG CCT CTT CTT EX-9-2-Cy GAA CTA CCT TGC CTG CTC CA EX-12-1-P TCT CCT TTT GGA TAC CTA GAT EX-12-2-Cy TGA GCA TTA TAA GTA AGG TAT

EX-13-1-P AGG TAG CAG CTA TTT TTA TGG EX-13-2-Cy ATC TGG TAC TAA GGA CAG

EX-14B-1-P TCT TTG GTT GTG CTG TGG CT 33 200538552

EX-14B-2-Cy EX16A-1P EX16A-2-Cy EX-18-1-P EX18-2-Cy Ex-19-l-Cy Ex-19-2-P

ACA ATA CAT ACA AAC ATA GT CTT CTG CTT ACC ΑΤΑ TTT GAC TAAT ACA GAC ATA CTT AAC G GG AGA AGG AGA AGG AAG AGT ATC TAT GAG AAG GAA AGA AGA GGC CAA ATG ACT GTC AAA GA TGC TTC AGG CTA CTG GGA TT ID NO .: 12 SEQ ID NO .: 13 SEQ ID NO .: 14 SEQ ID NO .: 15 SEQ ID NO .: 16 SEQ ID NO .: 17 SEQ ID NO .: 18

Table II mPCR Group II primers:

Ex-3-l-Cy Ex-3-2-P Ex-4-lP Ex-4-2-Cy EX-10-1-P EX-lO-2-Cy Ex-ll-lP EXll-2-Cy Int-19-l-Cy Int-19-2-P EX-20-1-P EX20-2-Cy EX21-1-P EX21-2-Cy

C GGC GAT GTT TTT TCT GGA GT ACA AAT GAG ATC CTT ACC C AGC TTC CTA TGA CCC GGA TA TGT GAT GAA GGC CAA AAA TG TGT TCT CAG TTT TCC TGG AT CTC TTC TAG TTG GCA TGC TT CAG ATT GAG CAT ACT AAA AG AC ATG AAT GAC ATT TAC AGC AA TCA TTC AGT GGG TAT AAG C CCT CCT CCC TGA GAA TGT TGG C TGG ATC AGG GAA GAA GG TCC TTT TGC TCA CCT GTG GT TGA TGG TAA GTA CAT GGG TG CAA AAG TAC CTG TTG CTC CA SEQ ID NO.:19 SEQ ID NO.:20 SEQ ID NO.:21 SEQ ID NO.:22 SEQ ID NO.:23 SEQ ID NO.:24 SEQ ID NO.:25 SEQ ID NO.:26 SEQ ID NO.:27 SEQ ID NO.:28 SEQ ID NO.:29 SEQ ID NO.:30 SEQ ID NO.:31 SEQ ID NO.:32

Main composition of PCR

20 // 1 reaction / sample: composition volume (// D 10XPCR buffer 2.0 25mM MgCl2 1.4 dNTPs (2.5mM) 4.0 primer mix (multiple 10x) 3.0 7¾ points DNA polymerase 0.6 ddH20 3.0 DNA 6.0 total volume 20 34 200538552 PCR cycle conditions Heating start 94 ° C 15 minutes 94 ° C 30 seconds, 60% ramp 60 ° C 30 seconds, 50% ramp 72 ° C 50 seconds, 35% ramp 72 ° C 8 minutes Use a Perkin Elmer 9700 30 cycles for each primer pair to determine the optimal primer concentration. The reaction volume can be adjusted according to the needs of the experiment. _ 户户 浚 里 · After the amplification reaction, the PCR product is a QIAqiuck PCR purification kit (QIAGEN, Cat # 281 04) or purified with exonuclease I treatment (Amersham). For the latter procedure: add 8 # 1 fraction (a 1 i quot) of PCR products to a clean and containing 2 5 // 1 In a test tube of exonuclease I (Amersham), incubate at 3TC for 15 minutes, and then denature it at 80 for 15 minutes. After that, single-stranded DNA can be produced by: PCR reaction product and 2.5 units of Exonuclease in 37 C culture in i-fold buffer 20 Minutes, followed by heating to 75. 匸 10 = bell to deactivate the enzyme. Under these conditions, the enzyme breaks down one strand of double-stranded DNA from the phosphorylated end and releases a 5-phosphorylated single nucleotide. t Little et al., 1967). Single-share targets can also be obtained through others. It is produced by a method known in the field of metaphysics, however, it is not desirable to heat the pcR material to generate single-stranded DNA. This single strand wake can be used directly in the array by 35 200538552. Distant cross-CFTP gene sequence obtained from Genebank & ww'ncJiijxij ^^ j ^ iji ^ g ^) was used as a wild-type pattern. The 52 probes were divided into two groups based on their sequence homology. They were based on "heuristic" and "algorithm". That is, in this method, different probes in the possible range are avoided. Overlapping homology of the needles. Mutants contained in each population were selected to reduce the heavy abundance between probe sequences in any population, and were therefore used to narrow the population within multiple analysis conditions-populations (intra-group) cross-hybridization. The probe sequence is designed by PRIMER 3.0 software (see this series as an appendix), so that each probe has the following characteristics ... (b) — the mismatch is located in the center of the probe (C) probe length is 6-21 bases; (d) low self-complementarity, (e) contains 30-60% GC content; and (f) no more than three consecutive identical bases. Each The probe sequence is aligned with its complementary exon sequence. See

lllt.p; //searrhlai]p.hprihrTri /, this series is an appendix. Calculate the ratio of virulence between each mold parental needle and non-desired target sequences (except for the mutation sequence in which the probe is to hybridize), and select each probe in the same array. The protozoan ratio of N between the protruding bismuth needle and the undesired target sequence is less than 50%. 36 200538552 * The selection of the needle is based on the error selection rules of the f test "into: eight as above. The probe selection can also be partially improved due to the experimental selection, and the test of the 4-knife consists of the test base pair containing the wrong pairing ( In particular, IG is stable. The probe can be correctly mixed with the error "; u pair of towels, and in some of its mules, the stable Λ-2 pairing can be avoided, or if the experiment proves When the incorrect hybridization can be reduced by using the antisense strand probe, the anti_sense strand probe is used ^ The sense strand probe is not used. The case of using the antisense strand probe is indicated in the list IU. Twenty-six CF-mutated wild-type and mutant probes were synthesized in sequence, and 5 'biotin-TEG or amine modification was performed at the 5' end (combined with the technology called Kamine a). Different bead chemistry can use a different At the 5, end, the biotin modification can be bound to the beads coated with neutravidin, and the amine modification can be bound to the beads coated with BSA. The probe system is dissolved in 1χ or deionized water at a concentration of 100 // M. For each type of bead, 100 # 1 containing 1% bead solids State solution system was 5〇〇 // i of TBS - Klx te, 〇.5MNaC12) washed three times. The probe was added to a 500 / M bead suspension and incubated in a roller at room temperature for 45-60 minutes. The beads were washed once with TBS-T solution (ΠTE, 0.15M NaCl2, · 05% Tween 20) or PBS-T (phosphate buffered saline, Tween 20), and TBS_2 (1X TE, 0.15M NaC 12 ) Wash once and resuspend in ΐχ TBS-2 solution. The beads are bonded to the wafer surface as previously described. The probes were also divided into two groups and assembled on two separate wafers. Combine a third group to perform a reflection test (including 5T / 7T / 9T polymorphism). Negative and positive control groups are also included on the surface of this crystal. The analysis signal is corrected using these control groups. For the negative control group, the beads were combined with 10-mer strands of dCTP (01 igo-C) and fixed on the wafer surface. For the positive control group, human / 3 actin sequences were used. The signal generated from 01 igo-C was used as the background value to subtract interference, and the / 3 actin system was used to normalize the value. Table IIIA-Hybrid population I Bead population mutation Bead population mutation 1 OLIGO-C (control group) 19 G542-M 2 BA 20 G551D-WT 3 OligoC-1 21 G551D-M 4 G85E-WT 22 R560-WT 5 G85E -M 23 R560-M 6 621 + 1G > T-WT 24 R553X-WT 7 621 + 1G > TM 25 R553X-M 8 R117H-WT 26 OLIGOC-3 9 R117H-M 27 1717-1G > A-WT 10 I148 -WT 28 1717-1G > AM 11 I148-M 29 3849 + 10kb-WT 12 A455E-WT 30 3849 + 10kb-M 13 A455E-M 31 W1282X-WT 14 508-WT 32 OLIGOC-4 15 OLIGOC-2 33 W1282X -M 16 F508 34 N1303K-WT 17 1507 35 N1303K-M 18 G542-WT 36 0LIG0C-5

38 200538552

Table IIIB-Hybrid Group II

Bead group mutation 1 BA 18 3659delC-WT 2 1898 + 5G-WT 19 3659delC-M 3 OLIGO-C (control group) 20 OLIGO-C-3 4 1898 + 5G-M 21 R1162X-WT 5 OLIGO -C-1 22 R1162X-M 6 R334W-WT 23 2789 + 5G-WT 7 R334W-M 24 2789 + 5G-M 8 1898 + 1G> A-WT 25 3120 + 1G > A-WT 9 1898 + 1G > A -WT 26 3120 + 1G> A-WT 10 1078delT-M 27 OLIGO-C-4 11 OLIGO-C-2 28 A455E-WT 12 D1152-WT 29 A455E-M 13 D1152-M 30 2184delA-WT 14 R347P-WT 31 2184delA-M 15 R347P-M 32 1078delT-WT 16 17 711 + 1G> T-WT 711 + 1G> TM 33 OLIGO-C-5 Table 3 me-hybrid groups in Cluster # (total 6 groups) Mutation 1 Stone muscle Kinesin 1 Oligo C 2 5T 3 7T 4 9T The probe sequences used to detect each mutation are as follows (such as the aforementioned probe selection for sense strand or antisense strand sequence):

Table IV Normal / variant sequence capture probes SEQ ID NO.:33 SEQ ID NO.:34 SEQ ID NO.:35 SEQ ID NO.:36 EX-3 AT GTT CTA TGG AAT CTT ΤΤ ΤΑ ΤΑΑ85E AT GTT CTA TGA AAT CTT ΤΤ ΤΑ

EX-4 ΤΑ TAA GAA GGT AAT ACT TC CT

621-M TA TAA GAA GTT AAT ACT TC CT 39 200538552

CC TCA TCA CAT TGG AAT GC AG CC TCA TCA CAC TGG AAT GC AG CAA GGA GGA ACG CTC TAT CG C CAA GGA GGA ACA CTC TAT CG C ATG GGT ACA TAC TTC ATC AA A ATG GGT ACA TAA TTC ATC AA A GAA TAT TTT CCG GAG GAT GAT GAA TAT TTT CCA GAG GAT GAT CAT TGT TCT GCG CAT GGC GGT CAT TGT TCT GCC CAT GGC GGT CT CAG GGT TCT TTG TGG TG TT CT CAG GGT TC TTG TGG TGG A TT ACA GTT GTT GGC GGT TGC TGG A GTT GTT GGA GGT TGC TGG AAA GAA AAT ATC ATC TTT GGT AAA GAA AAT ATC ATT GGT GT AAA GAA AAT ATC TTT GGT GT ATA TTT GAA AGG TAT GTT CT TT ATA TTT GAA AGA TAT GTT CT TT GAA ACA AAA AAA CAA TCT TTT GAA ACA AAA AA CAA TCT TTT TTG GAA AGT GAG TAT TCC ATG TTG GAA AGT GAA TAT TCC ATG ACT TCA TCC AGA TAT GTA AAA ACT TCA TCC TCC AGG TAT GTA AAA TAT AGT TCT TGG AGA AGG TGG TAT AGT TCT TTG AGA AGG TTA GCA AGG TGA ATA ACT TCT TTA GCA ACG TGA ATA ACT GAG TGG AGG TCA ACG AGC AAG GAG TGG AGA TCA ACG AGC AAG GTG GAG GTC AAT GAG CAA GA TGG TAA TAG GAC ATC TCC AAG TGG TAA TAA GAC ATC TCC AAG ACT GCA TAG ATG TGG ATA ACT CCA GCA T AC ATG TGG ATA GAA CTG TGA GCC GAG TCT TTA GAA CTG TGA GCT GAG TCT TTA TGG TTG ACT TGG TAG GTT TAC 3659 TGG TTG ACT TG TAG GTT TAC T TAA AAT GGT GAG TAA GA CAC 3849 T TAA AAT GGC GAG TAA GA CAC EX-20 TGC AAC AGT GGA GGA AAG CCT 1282X TGC AAC AGT GAA GGA AAG CCT EX-21 A TTT AGA AAA AAC TTG GAT CC N1303K A TTTAGA AAA AAG TTG GAT CC / 3 A-PROBE AG GAC TCC ATG CCC AG INT- 4 I148T EX-4 R117H EX-5 711 + 1G EX-7 334-M EX-7 347-M EX-7 1078DELT EX-9 A455E EX-10 F508 1507 EX-12 1898 + 1 Ex-13 2184delA EX-14B 2789 + 5G EX-16 31120 + 1G / A Ex-11 G542X EX-11 R560 EX-11-553 / 551 G551D R553X EX-11 1717-M EX-18 1152X EX-19-SENSE R1162X EX-19 INT-19 SEQ ID NO.:37 SEQ ID NO.:38 SEQ ID NO.:39 SEQ ID NO.:40 SEQ ID NO.:41 SEQ ID NO.:42 SEQ ID NO.:43 SEQ ID NO.:44 SEQ ID NO.:45 SEQ ID NO.:46 SEQ ID NO.:47 SEQ ID NO.:48 SEQ ID NO.:49 SEQ ID NO.:50 SEQ ID NO.:51 SEQ ID NO.:52 SEQ ID NO. : 53 SEQ IDNO.:54 SEQ ID NO.:55 SEQ ID NO.:56 SEQ ID NO.:57 SEQ ID NO.:58 SEQ IDNO.:59 SEQ ID NO.:60 SEQ ID NO.:61 SEQ ID NO.:62 SEQ ID NO .: 63 SEQ ID NO.:64 SEQ ID NO.:65 SEQ ID NO.:66 SEQ ID NO.:67 SEQ ID NO.:68 SEQ ID NO.:69 SEQ ID NO.:70 SEQ ID NO .: 71 SEQ ID NO.:72 SEQ ID NO.:73 SEQ ID NO.:74 SEQ ID NO.:74 SEQ ID NO.:76 SEQ ID NO.:77 SEQ ID NO.:78 SEQ ID NO.:79 SEQ IDNO.:80 SEQ ID NO.:81 SEQ ID NO.:82 SEQ ID NO.:83

40 200538552 The hybridization buffer was optimized for single and / or multiple hybridization analysis, and the composition was as follows (final concentration): 1. 125M tetramethylammonium chloride (TMAC), 18.75 μM Tris-HCl ( (PQ8 · 0), 0.75mM EDTA (PQ8 · 0) and 0.0375% SDS. A 10 // 1 hybridization mixture containing buffer and single-stranded DNA was added to the wafer surface, and it was incubated at 55 for 15 minutes. This is a short hybridization time compared to the normal number of heterosexuals with small η guards, because longer hybridization times lead to uncontrolled overhybridization. The wafer was washed three times with 1X TMAC buffer, then covered with a clean lid, and analyzed using the BAS imaging system. Analyze the images to determine the identity of each probe. The results are shown in Figures 1 and 2 below. Each dual gene line in the mutation was analyzed according to the following. First, the signals generated from the hybridized dual genes were corrected as follows: (·) Signals from dual gene A (calibrated with amplicons) = calibrated amplicon-hybrid original signal minus negative control group (background value) ) Original count (11) Signal of dual gene B (uncalibrated amplicon) = Uncalibrated amplicon miscellaneous debris, start signal minus original plan text φ of negative control group (background value), and then Calculate the ratio of dual genes: = dual ratio = signal of dual gene A / signal of dual gene B. When the value of ⑴ is less than or equal to 0, it is adjusted to 〇1 to avoid the display. Lines are described as homologous to the dual gene A (gene's polymorphism), and when the ratio of duality is < 0.5, the lines are recorded as intergenic (wild-type) with the duality. When the dual ratio is 0 8 to 12, it is recorded as heterogeneous by 41 200538552. Values falling between these thresholds are considered ambiguous and the analysis needs to be repeated. Example 11: Screening of multiple patient specimens-one-by-one comparison of hMAP by point rupture analysis method A number of patient specimens were obtained and enlarged for simultaneous screening. The amplification reaction method and probe design are as described above. After the amplification reaction, the analysis technique of the specimen is to compare the 2 6 CFTR mutation. One set of test systems was analyzed using the existing point-blot hybridization method, while the same sample was also analyzed using the method and reaction reagent of the present invention. The analysis results of each patient specimen were collected and compared. It was found to be 100% consistent in the two detection methods. For each mutation, those with a positive number of specimens are listed in Table V. Table V: The comparison test system of the test specimens via the point break method and the mutation #Positive values G85E 11 G85E / 621 + 1G 8 621 + 1G > T 11 621 + 1g > T / delF50 8 2 R117H 19 R117H / delF508 1 I148T 48 delF508 58 1507 11 delF508 / R560 1 G542X 44 G551D 11 R553X 15 1717-1G &A; A 14 Test 5 2 The value of French law # tn Ming 3 11 4 11

11 IX 24 1 5 5 15 4 11 11 42 200538552 R560T 3849 + 10kbC > T W1282X N1303K mPCR-WT 711 + 1G > T 711 + 1G > T / 621 + R334W R347P 1078delT A455E 1898 + 1G > A 2184delA 2789 + 5G > A 3120 + lg〉 A R1162X 3569delC D1152 mPCR-WT Total 5 3 1 2 5 3 3 5 7 118

9 3 11 1X 11 G 8 4 0 0 8 3 1X 0 / ^ 11 2 IX 11 7 11 11 ο ο ο ο 1X II 1X 11 80 9 6 6 7 8 031940081 6 0 3 3648213112323228589 It should be understood that the terms used in this article , Expressions, and examples are by way of illustration, not limitation, and procedures and methods can be performed in any order, unless the order of steps has been specified. The present invention is defined in the following patent application scope and includes all equivalents to the patent application scope. [Schematic description] Figure 1 shows the hybridization results of 29 different CFTR mutations. The smaller open bars show mutant crosses, and the "normal" hybrid lines show larger black bars (for example, : EX-1 0 has a high degree of mutant crosses). Fig. 2 shows the results of hybridization of 29 different CFTR mutations, which are different from those shown in Fig. 1. FIG. 3 shows the carrier of a negative control group of the array image after correction.

43 200538552 Background image.

44 200538552 Ordering fj table < 110 > Hashmi, Ghazala Seul, Michael

< 12〇 > Polymorphic hybridization analysis < 130 > hMAP < 150 60 / 470.806 < 151 > 2003-05-15 < 160 > 83 < 170 > FastSEQ for Windows Version 4.0 < 210 > 1 < 211 > 19 < 212 > DNA < 213 > artificial sequence < 220 < 223 > artificial primer < 400 > 1 gtcaagccgt gttctagat 19

< 210 > 2 < 211 > 23 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 2 gttgtataat ttataacaat agt 23

< 210 > 3 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 3 acttcaatag ctcagccttc 20 < 210 > 4 < 211 > 23 < 212 > DNA < 213 > artificial sequence 200538552 < 220 < 223 > artificial primer < 400 > 4 tatggtacat tacctgtatt ttg

< 210 > 5 < 211 > 18 < 212 > DNA < 213 > entry sequence < 220 > < 223 > artificial primer < 400 > 5 tggtgacagc ctcttctt < 210 > 6 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Human item < 400 > 6 gaactacctt gcctgctcca

< 210 > 7 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 7 tctccttttg gatacctaga t

< 210 > 8 < 211 > 21 < 212> DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 8 tgagcattat aagtaaggta t

< 210> 9 < 211 > 21 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > artificial primer < 400 > 9 aggtagcagc tatttttatg g 21 < 210 > 10 < 211 > 18 < 212 > ΠΝΑ < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 10 atctggtact aaggacag 18

< 210 > 11 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 11 tctttggttg tgctgtggct 20

< 210 > 12 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400> 12 acaatacata caaacatagt 20

< 210 > 13 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400> 13 cttctgctta ccatatttga c 21

< 210 > 14 < 211 > 20 < 212 > DNA < 213 > & engineering sequence-3- 200538552 < 220 > < 223 > artificial primer < 400〉 14 taatacagac atacttaacg < 210 > 15 < 211 > 20 < 212 > DNA < 13 >: artificial sequence < 220 > < 223 > artificial primer < 400 > 15 ggagaaggag aaggaagagt

< 210 > 16 < 211 > 21 < 212 > DNA < 213 > | Human Procedure ^ [< 220 > < 223 > Artificial primers < 400 > 16 atctatgaga aggaaagaag a < 21〇 > 17 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > — < 223 > artificial primer < 400 > 17 ggccaaatga ctgtcaaaga

< 210 > 18 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 18 tgcttcaggc tactgggatt < 210 > 19 < 211 > 20 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > artificial primer < 400 > 19 cggcgatgtt ttttctggag < 210 > 20 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Artificial primers < 400 > 20 tacaaatgag atccttaccc

< 210 > 21 < 211 > 20 < 212 > DNA < 213 > human process 刿 < 220 > < 223 > artificial primer < 400 21 agcttcctat gacccggata

< 210 > 22 < 211 > 20 < 212> DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400> 22 tgtgatgaag gccaaaaatg < 210 > 23 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 23 tgttctcagt tttcctggat < 210 > 24 < 211 > 20 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 >, artificial primers < 400 > 24 ctcttctagt tggcatgctt < 210 > 25 < 211 > 20 < 212 > DNA < 213 >. artificial sequence < 220 > < 223 > artificial primer < 400〉 25 cagattgagc atactaaaag < 210 26 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 26 acatgaatga catttacagc

< 210 > 27 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400〉 27 aatcattcag tgggtataag c < 210〉 28 < 211 > 21 <; 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 > 28 cctcctccct gagaatgttg g < 210 > 29 < 211 > 20 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > Artificial primers < 400 29 ctggatcagg gaagagaagg < 210〉 30 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > | Artificial primer <; 400 > 30 tccttttgct cacctgtggt < 210 > 31 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 >; artificial primer < 400 > 31 tgatggtaag tacatgggtg < 210 > 32 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial primer < 400 32 caaaagtacc tgttgctcca < 210〉 33 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223> artificial probe < 400 > 33 atgttctatg gaatcttttt a < 2 10> 34 < 211 > 21 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > artificial probe < 400 > 34 atgttctatg aaatcttttt a -column order nah 3521DN person > > > > 0 12 3 τ— τ— ^ — τ— 2 2 2 2 < 220 > < 223> Artificial probe < 400 > 35 tataagaagg taatacttcc t

< 210 > 36 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220〉-< 223 > artificial probe < 400> 36 tataagaagt taatacttcc t < 2i〇 > 37 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223〉 Artificial probe < 400 > 37 cctcatcaca ttggaatgca g < 210 > 38 < 211 > 21 < 212> DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400〉 38 cctcatcaca ctggaatgca g < 210〉 39 < 211 > 21 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 >; Artificial probe < 400 > 39 caaggaggaa cgctctatcg c

< 210 > 40 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 > 40 caaggaggaa cactctatcg c < 210 > 41 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 41 atgggtacat acttcatcaa a

< 210 > 42 < 211 > 21 < 212 > DNA < 213 > human hair train < 220 < 223 > artificial probe < 400〉 42 atgggtacat aattcatcaa a < 210> 43 < 211 > 21 < 212> DMA < 213 > Artificial sequence < 220 > < 223> Artificial needle picking < 400 > 43 gaatattttc cggaggatga t

< 210 > 44 < 211 > 21 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > artificial probe < 400 44 gaatattttc cagaggatga t 21 < 210 > 45 < 211 > 21 < 212> DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Probe < 400> 45 cattgttctg cgcatggcgg t 21 < 210> 46 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > artificial probe < 400 > 46 cattgttctg cccatggcgg t 21

< 210 > 47 < 211 > 21 < 212 > DNA < 213 > artificial sequence 丨 < 220 > < 223 > 丨 artificial probe < 400 > 47 ctcagggttc tttgtggtgt t 21 < 210 48 < 211 > 20 < 212 > DNA < 213 > artificial sequence — < 220 > < 223> artificial probe < 400 > 48 ctcagggttc ttgtggtgtt 20

< 210 > 49 < 211 > 21 < 212 > DNA < 213 > artificial sequence 10 200538552 < 220 < 223 >, artificial probe < 400 > 49 acagttgttg gcggttgctg g < 210 > 50 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 '& engineering probe < 400〉 50 acagttgttg gaggttgctg g < 210 > 51 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400> 51 aaagaaaata tcatctttgg t < 210 > 52 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > <; 223 > artificial probe < 400 > 52 aaagaaaata tcattggtgt < 210 > 53 < 211 > 20 < 212> DNA < 213 > artificial sequence < 220 > < 223 >; artificial probe — < 400 > 53 aaagaaaata tctttggtgt < 210> 54 < 211 > 22 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > artificial probe < 400> 54 atatttgaaa ggtatgttct tt 22 < 210 > 55 < 211 > 22 < 212> DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 > 5 5

atatttgaaa gatatgttct tt 22 < 210〉 56 < 211 > 21 < 212〉 D | iA < 213 > Λ sequence < 220 > r < 223> artificial probe < 400〉 56 gaaacaaaaa aacaatcttt t 21

< 210 > 57 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 >

< 223 > Artificial probe < 400 57 gaaacaaaaa acaatctttt 20 < 210 > 58 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 >. Artificial probe < 400 〉 58 ttggaaagtg agtattccat g 21

< 210 > 59 < 211 > 21 < 212 > DNA < 213 > artificial sequence 12 200538552 < 220 > < 223> Artificial probe < 400 59 ttggaaagtg aatattccat g 21 < 210 > 60 < 211 > 21 < 212 > DNA < 21 artificial sequence 1 < 220 > < 223> artificial_soil- < 400 > 60

acttcatcca gatatgtaaa a 21

< 210 > 61 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223〉 artificial probe < 400〉 61 acttcatcca ggtatgtaaa a 21

< 210 > 62 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 >

< 223> Artificial Probe < 400 > 62 tatagttctt ggagaaggtg g 21 < 210 > 63 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Probe <; 400 > 63 tatagttctt tgagaaggtg g 21

< 210 > 64 < 211 > 21 < 212> DNA < 213 > artificial sequence 13 200538552, 220 > < 223 > artificial needle selection < 4〇〇 > 64 tctttagcaa ggtgaataac t

< 210 > 65 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 > 65 tctttagcaa cgtgaataac t < 210 66 < 211 > 21 <21; 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe_ < 400 > 66 gagtggaggt caacgagcaa g < 210 > 67 < 211 > 21 < 212 > DNA < 213 > artificial Sequence < 220 > < 223> artificial probe < 400> 67 gagtggagat caacgagcaa g < 210 68 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe Needle < 400 68 gtggaggtca atgagcaaga < 210> 69 < 211 > 21 < 212 > DMA < 213 > Artificial Sequence 200538552 < 220 > < 223 > Artificial Probe < 400 > 69 tggtaatagg acatctccaa g 21 < 210 > 70 < 211 > 21 < 212 > DNA < 213 >, artificial sequence < 220 < 223 > artificial release needle < 400 > 70 tggtaataag acatctccaa g 21 < 210 > 71 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223〉 丨 Artificial release needle < 400 > 71 actccagcat agatgt ggat a 21 < 210 > 72 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 > 72 actccagcat acatgtggat a 21 < 210 > 73 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 > 73 gaactgtgag ccgagtcttt a 21 < 210 74 ·

< 211 > 21 < 212 > DNA < 213 > Artificial Sequence 15- 200538552 < 220〉. < 223 > Artificial Probe < 400 74 gaactgtgag ctgagtcttt a < 210 > 75 < 211 > 21 < 212 > hnja _ < 213 > human process to < 220 > < 223 > artificial probe < 400 > 75 tggttgactt ggtaggttta c < 210 > 76 < 211 > 20 < 212 > ΠΝΑ < 213> & Working sequence < 220 > < 223 > Artificial probe < 400> 76 tggttgactt gtaggtttac

< 210 > 77 < 211 > 21 < 212 > DNA < 213 > AX ^ IJ < 220 > __ < 223 > Modular probe < 400 77 ttaaaatggt gagtaagaca c

< 210 > 78 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > artificial probe < 400 > 78 ttaaaatggc gagtaagaca c.

< 210 > 79 < 211 > 21 < 212 > DNA < 213 > artificial sequence 200538552 < 220 > < 223 > human:] ^ 罙 针 &400; 79 tgcaacagtg gaggaaagcc t 21 < 210 > 80 < 211 > 21 < 212 > DNA _ < 213 > artificial sequence-~ < 220 < 223 > artificial probe < 400 > 80

tgcaacagtg aaggaaagcc t 21

< 210 > 81 < 211 > 21 < 212 > DNA < 213 > artificial sequence — < 220 > < 223 > artificial probe < 400 > 81 atttagaaaa aacttggatc c 21 < 210 > 82 < 211 > 21 < 212〉 DNA < 213> artificial sequence < 220 >

< 223 > Artificial probe < 400 > 82 atttagaaaa aagttggatc c 21 < 210 > 83 < 211 > 16 < 212 > DNA < 213 > artificial @ 列 < 220 > < 223 > artificial probe— < 400 > 83 aggactccat gcccag 16 -17-

Claims (1)

200538552 The scope of patent application: ▲ i.- A method for optimizing heterosexual analysis for detecting known gene mutations and polymorphisms, which is initiated by the following steps:,) Provide-group Oligonucleotide primer pairs, each of which has the ability to adhere to a complementary polynuclear penetrating strand to delineate a corresponding target region that contains at least one selected mutation or polymorphic region; b) the A set of oligonucleotide primer pairs is contacted with the target under conditions that can form an amplicon pair, and each amplicon pair contains a selected amplicon corresponding to the target sense or antisense strand Significant strands' and-amplicons correspond to the amplicon antisense strands of another target strand (meaningful or antisense target strands); C) Selecting a second family of coding probes, which have probes with different codes With different nucleotide sequences, the selected sense strand probe is an antisense strand or a subsequence (meaning an antisense strand of a complementary amplicon), And other antisense strand amplicons mean "non- Selected amplicons) All or substantially complementary probes, and the selected antisense strand probes enable each of the antisense strand probes and the sense strand of the amplicon or its _ subsequence (meaning " Complementary amplicon sense strands, and other sense strand amplicons mean "non-selected amplicons,") all or approximately complementary; and wherein the method includes: using a sense strand probe and complementarity The antisense strand of the amplicon is separated in more than one different container to reduce the cross-hybridization between the probe and the non-selected amplicon in order to separately operate the hybridization reaction in different containers. The number of the container It should be as few as possible, so that the sequence similarity between the expander (2005) and the probe (in the same probe) in the same container does not exceed the preset acceptable level. 〇 2. If the method in the first scope of the patent application, Wherein, the polynucleotide is mRNA, cDNA or a double-stranded polynuclear malate containing DNA. 3. The method according to item 1 of the patent application, wherein the probes having different sequences are obtained by combining the probes with a carrier (including small Beads) carrier binding Code, the carrier has different optical identification marks. 4. If the method of the scope of the patent application is No. 3, the code is colored. 5. If the method of the scope of the patent application, No. 1, the primer system in one primer pair A calibration mark is marked at the 5 ′ end, and the other primer in the primer pair has a maleic acid modification at the 5 end. 6. The method according to item 5 of the patent application scope, wherein the amplicon incorporating the phosphate modified primer It is decomposed. 7 · The method of claim 1 or 2 in which the scope of the patent is declared, wherein the hybridization between the amplicon and the probe is determined by a calibration mark that detects the binding from the amplicon. 8 · — a kind of An optimized hybridization analysis method for detecting known genetic mutations or polymorphisms, which is initiated by the following steps: a) Provide a set of oligonucleotide primer pairs, each primer pair has a complementary complement The ability of the polynucleotide strand to bind to delineate the corresponding target region or the target region containing at least one selected mutation or polymorphism; b) the group of oligonucleotide primer pairs with the target can be Amplicon pair formation Contact under conditions' each amplicon pair contains-selected expansion 46 200538552 amplicon meaning stock (corresponding to the underlying meaning stock or antisense stock) and an amplicon antisense stock (corresponding to other underlying stocks (meaning Or antisense target strand)); C) Select a second family of coded probes, in which probes with different codes have different nucleotide sequences, and the selected sense strand probes are Sense strand or primary sequence (subsequence) all or approximately complementary probes (meaning an "antisense strand of a complementary amplicon, and another antisense strand amplicon means" non-selected "", And the antisense strand probes are selected so that each antisense strand probe can be fully or substantially complementary to the sense strand or subsequence of an amplicon (meaning a "complementary expansion" '' Zhengluzi sense strand '', and other sense strand amplicon means `` non-selected amplicon, ''): and wherein the method includes: the amplicon maintains sequence similarity and the probe and non-selected amplicon Degree of parental cross Below a preset acceptable level, it is to replace the sense strand probe with one or more antisense strand probes (and replace the amplicon antisense strand with a complementary amplicon sense strand). 9. The method of claim 8 in the scope of the patent application, wherein the alternative antisense strand probe can be fully or substantially complementary to the alternative sense strand probe. 10. The method as claimed in claim 8 wherein the polynucleotide is an mRNA 'cDNA or a double-stranded nucleotide containing DNA. 11. The method according to item 8 of the patent application, which allows probes with different sequences to be encoded by combining the probes with a carrier (including beads), which carriers have different optical identification marks. 1 2. As stated in the method of item iJ of the patent scope, wherein the coded band 47 200538552 is colored. 13. The method according to item 8 of the scope of patent application, wherein the -primer in the -primer pair is calibrated at the 5 'end and the other primer in the primer pair has a phosphate modification at the 5'end. U. The method according to item 13 of the application, wherein the amplicon line combined with the acetic acid-modified primer is decomposed. 15. The method according to item 8 of the scope of patent application, wherein the hybridization line between the amplicon and the probe is determined by detecting a calibration mark associated with the amplicon. 16. An optimized hybridization analysis for a known genetic mutation or polymorphism, including the following steps: a) Provide a set of oligonucleotide primer pairs, each primer pair has a complementary The ability of polynucleic acid strands to adhere to describe the corresponding target region, which contains at least one selected mutation or polymorphic region; b) the set of oligonucleotide primer pairs and the target can form The amplicon pairs are contacted, and each amplicon pair contains a selected amplicon sense strand (corresponding to the target sense strand or antisense strand) and an amplicon antisense strand (corresponding to another Target stocks (meaning or antisense target stocks)); and where the degree of sequence similarity and cross-hybridization and cross-hybridization with partially complementary non-selective wipes are below a preset acceptable level, The number of amplicon regions that hybridize to the probe via 4 疋 is controlled. 17. The method of item 16 in the scope of patent application, wherein the number of amplicon regions selected as the hetero-parents of the tadpole probe is determined, and amplicons smaller than the above regions are generated in a subsequent step. 48 200538552 18. A method for designing a needle array for hybridization analysis for detecting gene mutations or polymorphisms, which is initiated by the following steps: a) Provide a set of oligonucleotide primer pairs, each Primer pairs have the ability to adhere to complementary polynucleotide strands to describe the corresponding target region or region containing at least one selected mutation or polymorphism region; b) recruit the group The nucleotide primer pair can form amplification with the target. Each contact is made under the condition of 1 and each amplicon pair contains a selected amplicon sense strand (corresponding to the target meaning strand or antisense strand) and an amplicon antisense strand (corresponding to another target strand (meaning Or antisense stock)); φ ”c) Select the second group of coding probes, in which bismuth needles with different codes have different nucleotide sequences, and the selected sense probes are compatible with Antisense strand or subsequence probes that are all or substantially complementary (meaning an antisense strand of a "complementary amplicon," and other antisense month amplicons mean "non-selected Amplicons, and), and the selected antisense strand probes allow each antisense strand probe of the amplicon to be fully or substantially complementary to the sense strand or subsequence of an amplicon ( Means a "complementary amplicon sense strand, and other sense strand amplicons mean" non-selected amplicons "); and wherein the method includes the following steps: a) detecting the individual sense strands Degree of homology between probes or individual amplicon sense strands b) The number of probes of the sense strand is divided into one or more spoons, and the sense strands of the complementary amplicon are divided into corresponding groups, and a knife method is implemented in order to maintain a gap between the mother group of probe groups and one strand. The sequence similarity of 49 200538552 # 同 %% is maintained at the degree of similarity between the parent-group amplicon meaning strands) so that it is below a preset acceptable level; C) Implement the following steps ... ⑴When walking in the hybrid material Tit, it is decided that ―the amplicon's… corresponding to the corresponding ㈣ group; or, when contacting under hybridization conditions, = needle group—the strands corresponding to the corresponding amplicon group, Whether the degree of non-selected one-sixth and inter-parents of the unit and the probe set or side amplicon group exceeds a preset acceptable level; and if not: Α (ϋ) is in a separate group 'Maintain the strand of the amplicon and one strand of the probe set' ^ Repeat steps (A) (i) for the other strand of the amplicon or the straight strand of the probe set (A) (i); (B) (υ but assuming that the degree of cross-breeding does not exceed the acceptable level; 'replace the cross within the individual group The hybridized probe and a complementary antisense strand probe, or the cross hybrid antisense strand amplicon and a complementary amplicon sense strand; and (B) (ii) the replaced probe and the amplified Repeater step (A) (0), assuming that the degree of cross-hybridization does not exceed the acceptable level: within its individual group, maintain the antisense strand probe and the selected anti-strand amplification -A strand, and the other strand of the amplicon group at the same time (A) (i); (B) (iii) But suppose that the replacement probe and amplicon are repeated in step (A) (i ), The degree of cross-hybridization is higher than this acceptable degree: decide whether to contact with the probe in any other group, or the amplicon in any other group, according to the replaced probe and the amplicon respectively, such as When applicable, 50 200538552 the degree of the cross-breeding does not exceed the acceptable level; and it remains within the individual group of the permutation; but assuming that according to the method of decision: An acceptable degree, replacing the replaced strand into a new group, and (C) the amplification When the probe or an other group, repeating steps (A) (i) to (B) (iii), as applicable. 19. The method according to item 18 of the patent application scope, further comprising the steps of: providing conditions for generating a subgroup for each of the set of amplicon sense strands and amplicon antisense strands (respectively Is "bamboo," and "MP ,," wherein the sub-population has one or more different regions in the sequence, and the amplicons of a WT sub-population are relative to one of the genomic sequences. Wild-type region, and the amplicons in the WT subgroup are relative to a mutation or polymorphic region in the genomic sequence; four subgroups of the probe are selected (named as WT meaning unit, antisense, respectively) Stocks, MP meaning stocks, MP antisense stocks), so that the probes in each population can be relative to the amplicon meaning stocks, which are completely complementary or substantially complementary in the subgroups or sequences that are calibrated but complementary; Decide ... (i) Between a WT amplicon antisense strand and the substantially complementary Mp sense strand probe 'or a Mp & sense strand amplicon and the substantially complementary WT sense strand probe Whether the degree of cross-hybridization will exceed The acceptable degree is assumed to be (ii) the interaction is determined when the MP meaning stock or the antisense stock is replaced with a complementary WT antisense stock probe or a complementary MP antisense stock probe, respectively. Whether the degree of hybridization will fall within the acceptable range of 51 200538552; and if the county version is sighed, (Hi) decides whether the WT antisense stock is lighter than the Li Er antisense probe, if applicable At the same time, for cross-hybridizing the MP amplicons or other amplicons with other meanings, the amplicons with the meaning of rr will be respectively acceptable, and jealousy < θ, w exceeds and false cross疋, (v) decide whether to replace the WT strand probe or the mp antisense strand um, I Jiuyi Yue again, and together with the complementary MP to expand the WT amplicon to a separation from 纟 + π 5 If there is a long-standing group of Igang, if applicable, it will be common to any set of other subgroups in the separated subgroups, ^ octabutase probes and amplicons from steps (i) to (lv) 'and The cross-hybridization of other probes and amplicons in the separated sub-populations will exceed the acceptable level of β. Separated within the ^ knife ^ sub-group; but suppose that ⑺ repeat step (iv) using other points: gang's confidant monarch '' and carry on the succession of the probe amplicon succession '' to this period and the separation Subgroups until acceptable levels are met. ^ 2 20. The method of claim 18 or 19, wherein the determination of the acceptability includes reducing or reducing the number of G-T base pairings. 21. The method of claim 18 or 19, wherein the accessible degree is determined by the computer program PAMtm. ^ M. The method according to item 8 of the patent application, wherein the subject of the polynucleotide is an mRNA, cDNA, or a double-stranded polynucleotide comprising DNA. 23. The method of claim 18, wherein the probe has a different sequence from the probe bound to a bead-containing carrier, and the carrier has a different optical identification mark. The method of applying the scope of patent application of the two-color-colored eruption 'its + where the monument is 25. The method of applying the patent scope No. 18 or 19, in which one primer in a 200538552 sub-pair is at 5, end calibration and calibration Mark, and the other primer in the primer pair has a monophosphate modification at the 5, end. 26. The method according to claim 25, wherein the amplicon line incorporating the phosphoric acid-modified primer is decomposed. 27. The method of claim 18 or 19, wherein the hybridization between the amplicon and the needle is determined by the calibration mark combined with the amplicon. 28. The method of claim 25 in the scope of patent application, wherein the calibration mark It is Cy3, Cy5 and Cy5.5. The method described in item 29 of the same patent application, where the WT probe and the MP probe are very similar in sequence, but only one nucleotide position is less than that in the same application. The method of item 1, in which the Mτ amplicon MP probes are very similar in sequence, only at a position of 3 nucleotides which is not 3 i · — according to the patent application The probe or amplicon selected by the method 32 ·-a probe for selecting 选 related to cystic fibrosis with n 'has a sequence of SEQ ID Nqs. 33 to 83. y 33. A mutation or polymorphism at a test locus uses a vector-array of display probe pairs, complex λ, #r ^, and eight have different vectors that can not be targeted at Chengbei, in order to detect Targeting-hybridization method used to identify the normal dual gene of Jingjing-can be used to identify the corresponding solid, two-sided mutation pair by hybridization method 53 200538552 because 'the vector is encoded for identification display The probe includes: from a specimen suspected of being mutated, you use a pair of primers in the region of the mother and amplify the genomic region related to the dual gene of the ^ ^ 疋-Each line was calibrated at the 5 'end to generate a single amplicon; 、, 且, and 工 bismuth 疋 produced a single-strand amplicon; the probe pair displayed on the vector was placed on a substrate; intrinsic Miscellaneous 4 does not exceed the time required for the hybridization between the & needle and the amplicon. M 2 pieces will contact the probe pair array and the amplicon; the amplicon detection probe and the amplicon ^ ^ ^, Spread fathers, based on the The probe array _ crosses the signal of the amplicon; and decodes the array to identify the corresponding mutation or polymorphic amplicon of f q A, thereby determining that _3 is 4 ', ^^ / _, and the 33rd In the method of this item, the M strand amplicon line is produced by digesting one strand of the amplicon. 35. If the scope of the patent application is No. 34, phosphorylation is used, and one of the amplicon strands 3R is preselected by the primers for digestion. 36. The scope of the patent application is based on the introduction of endonucleases. The law, the method of application of the digestion application special scope item 33, is located in the CFTR area. And woocus) 38. The probe pair according to the scope of the patent application is fixed on the substrate. ">", Where the carrier exhibits the method of claim 33 of the patent patent, wherein the reaction time 54 200538552 does not exceed 15 minutes. 40. The method according to claim 33, wherein the carrier is a microbead. 41. For example, the scope of the patent application: item 33 *, wherein the matrix and the combined carrier can be inspected under a microscope. 42. The method of claim 33, wherein the genomic region is mRNA (or a cDNA derived therefrom) or a double-stranded polynucleic acid containing μα. 43. The method of claim 38 in the scope of patent application, wherein the microbeads are encoded with different optical marks. 44. The method according to scope 33 of the application for a patent, wherein the compilation is colored. 45 · —A method for distinguishing homozygote, heterozygote and wild type (used for mutation or polymorphism or wild type dual gene in the target sample), which uses the results obtained from the probe array, which is designed Used to detect the specified mutation or polymorphic dual gene and wild-type dual gene through the hybridization of the probe and the target, wherein the result includes the mismatched probes—standard, ,,, and ° a generations. g Compensation, which includes: Enlarging the genomic region of the target specimen (the assay system is predicted to contain the selected canine or polymorphic dual gene or the corresponding wild-type dual gene) to produce a calibration corresponding to Amplicons of selected mutations or polymorphic dual genes ('' canine / polymorphic amplicons, '') and calibrated repeats corresponding to wild-type dual genes (wild-type amplicons ,,) Daughter vector amplicons; an array of probe pairs is provided, where one strand is complementary to a mutant / polymorphic 55 200538552 amplicon and the other strand is complementary to the corresponding wild type amplicon; making the array For contact with the amplicon; / wild-type and mutant / polymorphic amplicon binding, based on the signal from the calibrated binding amplicon 'This signal can be corrected by adjusting the mismatch hybridization in the following ways (1) Determine the signal strength from mutation / polymorphic amplicon and wild type amplicon hybridization, correct the background signal, (⑴ & determine the proportion of this signal (ie, ⑹: mutation / polymorphism) Intensity or ratio to wild type (b): Intensity of wild type to mutation / polymorphism); and to set three numerical correlation ranges for this ratio: (i) where the lowest range of ratio (a) represents the inspection system Is a wild-type homozygote, and the lowest range of the ratio (b) indicates that the specimen is a mutant / polymorphic homozygote, (Π) the median range is expressed as an heterozygote, and (iii) the ratio of (a) The highest range indicates that the test system is a mutant / polymorphic homozygote, and the highest range of the ratio (b) indicates that the test sample is a wild-type homozygote. 46. If the method of 45 items of the patent application, further includes Wild type and mutation / polymorphism The step of generating single-stranded DNA in the booster. 47. If the method of item 46 of the patent is requested, further comprising the step of calibrating one of the wild type or mutation / polymorphism. 48. If the item of patent scope is 45 The method in which the ratio is explained as follows: > 2 represents mutation / polymorphic homozygotes, < 0.5 represents wild type homozygotes, and 0.8 to 1.2 represents heterozygotes. 49. A correction Method for false positive signal from wrong paired probe-sample (or probe-amplifier) binding, based on the signal obtained from the column of the oligonucleotide probe array 56 200538552, r ^., ° In addition, the probe passes through probe hybridization or probe pairing to generate self-expansion expansion needles, including heterozygous fathers, to detect genomic mutations or polymorphisms to form an array of probes; Place the array and specimens (or the array and pongs) in contact with each other at a temperature of approximately 5%, and then let them come in contact with each other; after passing a number of set temperature points, "," Indicates that there is a temperature at which a particular mismatched hybrid will be expected to de-adhesive; an signal generated from the array is monitored during heating to determine the starting heating point and the number of hybrids at the set temperature point (Or the relevant number and solution of the monitoring step results, which are based on the assumption that in any different setting, any signal is from the wrong pair. The wrong pair is expected to be below each set point. De-bonding. 50. For example, the method of 49 patent applications, where the signal is Autocorrelation to calibration on amplicons or specimens. 51. The method of claim 50 in the scope of patent application, wherein the calibration is optically detectable. 52. The method according to item 49 of the patent application, wherein the specimen or amplicon in the mismatched heterofather has a nucleotide in the sequence that is different from the correctly paired specimen or amplicon. 53. The method according to item 49 of the patent application range, wherein the temperature range is from 45 to 60 ° C. 54. A method of designing a probe array for use in complementary amplicons 57 200538552 Hybrid analysis includes: Detecting mutations and polymorphisms known in the genomic region (i) Φζ for an amplicon Jiajing, one of which was designated as Stupid Moon a, and its complementary strand was designated as antisense strand, the amplicon line ... ▲, and the specific genomic region of polymorphism; ) Select an amplicon from the family; (iii) Align the selected amplicon 9 with the remaining amplicons in the Dai family, which is arranged by pairing or multiple strokes The homology score of the daughter; the sequence arrangement of the reed, and the selection of the selected amplification; the family is arranged according to the increasing or decreasing homology score ... the son; (V) removes the homology score from the family Amplicons that exceed the preset acceptable range, and place the removed amplicons (and complementary probes) in the subgroup :, and repeat steps ⑴ to (for other amplicons in the family) V) (VI) Place each amplicon in turn in contact with alder needles in a particular group and decide to cross-hybridize with other probes in that group; "(Vii) select any probe and expand The complementary strand of the proton (the cross-hybridization of step (vi) 7 exceeds a preset acceptable level), and again decides to cross-hybridize with other probes in this group; (VI 11) In step (vi i) Any probes and amplicons that cross-hybridize beyond a preset acceptable level are placed in a separate population. 55. The method of claim 54 in which the method is repeated after step (vi U), but The goal is to generate the smallest number of amplifications. Ethnic groups. 5 6. For the method of applying for patents, the method of item 54 in Si, wherein after step (V111), Mugu, Xi, Xi; a predetermined number of amplicons (and probes): pre: Γ formula , And determine whether the group is based on the definition-the new lower most homologous homology score to determine which amplicons (and probes) need to be placed in the-new and separated group. · If applied: Method of 54 items of patent scope, which is generated by "The son reduces the parental cross-hybridization, the amplicon is shorter than the amplicon that exhibits excessive cross-hybridization. The method of experimental image of the positive array signal, which is derived from: re-hybridization-intermediate analysis, Individual signals in represent hybridization events' and where the optically encoded vectors are used to encode the individual hybridization events in this experiment, including: the use of vectors from negative control groups (ie, vectors that are encoded but not related to hybridization events) The signal constructs a background image; and the background image signal is subtracted from the "type" test image to generate a corrected test image. 59. The method according to item 58 of the scope of patent application, wherein the boxed (ie, non-variable in space) area of the background image is subtracted from the test image, and then the test image is divided by the corrected background image . 60. The method of claim 58 in which the background image is obtained by positioning the center of the negative control group vector contained in the array in a pre-selected area. The negative control group vector is coded and designed such that The Voronoi board that does not participate in the complex and construction is composed of a series of polygons 59 200538552, each containing a negative control group carrier, and each polygon is filled with the strength of the constructed negative control group carrier to generate image. 61. The method of claim 58 in which the filtering operation is applied to correct for the influence from a nearby negative control group carrier. Pick up, schema: as the next page 60