WO2004038042A1 - Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn - Google Patents

Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn Download PDF

Info

Publication number
WO2004038042A1
WO2004038042A1 PCT/CN2002/000751 CN0200751W WO2004038042A1 WO 2004038042 A1 WO2004038042 A1 WO 2004038042A1 CN 0200751 W CN0200751 W CN 0200751W WO 2004038042 A1 WO2004038042 A1 WO 2004038042A1
Authority
WO
WIPO (PCT)
Prior art keywords
temperature
gene chip
nucleic acid
detecting
double
Prior art date
Application number
PCT/CN2002/000751
Other languages
English (en)
French (fr)
Inventor
Ben Gao
Original Assignee
Ben Gao
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ben Gao filed Critical Ben Gao
Priority to CA002429101A priority Critical patent/CA2429101A1/en
Priority to JP2004545671A priority patent/JP2005519642A/ja
Priority to US10/380,112 priority patent/US7101671B2/en
Priority to AU2002338167A priority patent/AU2002338167A1/en
Priority to PCT/CN2002/000751 priority patent/WO2004038042A1/zh
Priority to EP02772012A priority patent/EP1437416A4/en
Priority to CNB028022254A priority patent/CN1164769C/zh
Publication of WO2004038042A1 publication Critical patent/WO2004038042A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the invention relates to a gene detection technology, in particular to a method and a device (G01N33 / 50) for detecting a nucleic acid hybrid pair on a gene chip by using a four-dimensional parameter. Background technique
  • Watson and Crick et al Proposed the concept of a double-helix structure model of DNA. They point out: (1) A DNA molecule is made up of two parallel polynucleotide strands in opposite directions, and these two strands have chemically opposite directions. (2) There are certain rules for base pairing: Chargaff et al. Used chromatography to analyze the base composition of various biological DNA and found that the number of adenine (A) and the number of thymine (T) in the DNA are equal. The number of cytosines (C) is equal to the number of guanines (G). Therefore, there are four possible base pairs in DNA: A-T, T-A, G-C, C-G.
  • each chain can have any base sequence, but due to the regularity of base pairing, if the base sequence of one chain is determined, the other chain must be There is a corresponding base sequence.
  • hybridization Since the double helix structure of DNA is mainly maintained by hydrogen bonds and hydrophobic bonds, all factors that can destroy the hydrogen and hydrophobic bonds such as heating, acid-base, and organic solvents can cause denaturation, making the double 'helix structure of DNA into an irregular line. group.
  • the "renaturation" of different ti DNA fragments by complementary base pairing is called hybridization. Hybridization can occur not only between DNA and DNA strands, but also between homologous sequences of DNA and RNA strands. During the hybridization process, two complementary single-stranded DNAs form a double-bond hybrid in a non-covalent manner. When the sequence of one of the strands is known, by detecting the hybridization process, it is possible to find out whether the unknown D N A sample contains the presence of D N A complementary to the known sequence.
  • nucleic acid hybridization is a process in which two complementary single-stranded DNAs form a double bond hybrid by non-covalent bonding. Can be used to determine whether an unknown DNA sample contains DNA complementary to a known sequence. The most common method is to immobilize a gene of a known sequence on a solid support, and then use it to complement the complementary sequence in solution Oligonucleotides are hybridized to enable specific DNA detection in the liquid phase.
  • DNA hybridization reactions require the use of labeling methods to detect hybridization signals. These methods allow in situ detection and can be highly sensitive. For example: The detection limit of PCR technology can reach nmol / 1; DNA computer technology also provides a method to detect a specific DNA sequence from a large number of mixed systems; Due to the application of short-wave fluorescence and confocal microscope technology, fluorescent labeling has become a detection Trace DNA is very sensitive and commonly used method.
  • the gene chip (DNA chip, or gene microarray) device only includes three-dimensional parameters of XYZ, for example, GeneCMp®, a gene chip produced by Affymetrix in the United States. In these gene chips, oligonucleotide DNA probes are usually fixed on glass substrates.
  • the base arrangement and chain length of oligonucleotide DNA probes can be characterized by three-dimensional parameters of XYZ.
  • the single-stranded polynucleotide target sequence is directly labeled with a fluorescent dye, and then hybridized with the chip array probe to form a double strand.
  • the fluorescence intensity of the chip array is detected by a scanner to obtain sample information.
  • U.S. patents US-5,445,934 and US-5,744,305 disclose gene chip devices containing a large number of DNA detection sites manufactured using photolithographic synthesis technology. Among these devices, all of the devices are related to oligonucleotide probes and other DNA probes fixed on the chip. The hybridization of the needle sequences is performed simultaneously at the same temperature.
  • oligonucleotide probes have different chain lengths and base composition (GC content), their melting temperatures (Tm)-that is, 50% of the probe and target nucleic acid strands The temperature at time of departure is also different, so the optimal hybridization temperature must be different. Due to the disadvantages of inconsistent hybridization temperatures, the accuracy of the results cannot be guaranteed and single base mismatches cannot be identified.
  • US Patent No. 6,238,868 discloses a microchip hybridization array with electric field as a free parameter, which speeds up the basic process of hybridization. However, its manufacturing is complicated, the cost is high, and the sample is polynucleoside.
  • the acid target sequence needs to be fluorescently labeled or hybridized with another fluorescently labeled reporter probe, which cannot guarantee the correctness of the results of the high-throughput full-array chip, which limits the widespread application of array hybridization.
  • the purpose of the present invention is to provide a method for detecting a nucleic acid hybrid pair on a gene chip with a four-dimensional parameter.
  • the method is based on the traditional detection of a nucleic acid hybrid pair using three-dimensional parameters of XYZ, and a temperature is introduced as a fourth-dimensional parameter for scanning.
  • the melting temperature of the fluorescent label of the sample-like nucleic acid hybridization pair changes with the temperature rising to the melting temperature to obtain the melting temperature.
  • the comparison of the melting temperature of the standard nucleic acid hybridization pair to determine the single-stranded nucleotide The method is simple, accurate, sensitive and specific.
  • Another object of the present invention is to provide a device for detecting a nucleic acid hybridization pair on a gene chip with a four-dimensional parameter.
  • the device is simple to manufacture and low in cost, and can ensure the correct result of the high-throughput full-array chip even if the hybridization temperature of the gene is not consistent. Sex.
  • the object of the present invention is to be achieved by the following measures: 'A method for detecting a nucleic acid hybridization pair on a gene chip with a four-dimensional parameter, including the following steps:
  • the melting temperature Tm of the double strand is obtained from the peak center of the curve. By comparing this melting temperature Tm with the melting temperature of a known double-stranded hybrid double-stranded strand, the characteristics of the single-stranded polynucleotide sequence of the sample hybridized with the probe of the gene chip can be detected.
  • the above (2) further includes a step of washing away the excess sample polynucleotide chain and the reaction solution in the device with a buffer solution containing no fluorescent dye and at the same temperature as the annealing temperature.
  • the annealing temperature Th is 4-89 ° C.
  • the melting temperature Tm is 8-100 ° C.
  • the temperature increase rate is o.oi-rc / second.
  • the reaction solution containing the double-strand mosaic fluorescent dye is selected from one of SYBR Green I reaction solution, SYBR Green II reaction solution, and SYBR Gold produced by Molecular Probe.
  • Another object of the present invention can be achieved by the following measures:
  • a device for detecting a nucleic acid hybridization pair on a gene chip with a four-dimensional parameter comprising a container capable of accommodating a gene chip, a gene chip, a temperature-controlled thermal cycling device, and a liquid inlet and a liquid outlet provided on the container; A gene chip is placed in the container, and the container is connected to a temperature-controlled thermal cycling device.
  • the temperature-controlled thermal cycle device includes a temperature sensor, a thermal cycle device, and a temperature control device; wherein the temperature sensor is connected to the temperature control device, and the temperature control device is connected to the thermal cycle device.
  • the object of the present invention is also to provide a device for detecting a nucleic acid hybridization pair on a gene chip with a four-dimensional parameter.
  • a four-dimensional parameter detection device for detecting a nucleic acid hybridization pair on a gene chip is used for detection and separation of a DNA sample.
  • a four-dimensional parameter detection device for detecting nucleic acid hybridization pairs on a gene chip is used for detection and separation of RNA samples.
  • the method of the present invention is based on the traditional detection of nucleic acid pairs using three-dimensional parameters of XYZ, and introduces a temperature as a fourth dimension parameter to scan the fluorescence intensity of the fluorescent marker of the nucleic acid hybridization pair as the temperature rises to the melting temperature.
  • the melting temperature can be obtained by comparing the melting temperature with the standard nucleic acid hybridization pair to determine the characteristics of the single-stranded nucleotides of the hybridized sample. This method is simple, accurate, sensitive, and specific.
  • the present invention introduces a temperature scanning method, which solves the problem of whether the result is correct due to inconsistent hybridization temperature of the chip array probe, and provides simple and clear criteria for the determination of the yin and yang of the result.
  • sample polynucleotide target sequence in the method of the present invention does not need to be labeled, nor does it need to use another fluorescently labeled reporter probe, which is convenient for operation.
  • the device of the present invention has a simple structure, can be used with a variety of commercial chips, and has a low cost; even if the hybridization temperatures of the genes are inconsistent, the accuracy of the high-throughput full-array chip results can be guaranteed.
  • Fig. 1 is a schematic structural view of the device of the present invention.
  • FIG. 2A is a melting curve (F ⁇ T) obtained by scanning a gene chip array probe point in the method of the present invention
  • FIG. 2B is the derivative melting curve (dF / dT ⁇ T) obtained by scanning the probe points of the gene chip array in the method of the present invention, the best way to implement the present invention
  • the device 9 includes a transparent glass box 1, a commercial gene chip 2, a temperature-controlled thermal cycler 3, and a liquid inlet 5. And a liquid outlet 7.
  • the temperature-controlled thermal cycling device 3 includes a temperature sensor 4, a thermal cycling device 8 and a temperature-control device 6; the temperature-control device 6 is a temperature-control computer; wherein the temperature sensor 4 is connected to the temperature-control device 6, and the temperature-control device 6 is connected to a thermal cycler 8.
  • the temperature control computer 6 controls the temperature of the reaction solution and the gene chip 2 in the glass box 1 through the temperature sensor 4 and the thermal cycler 8 according to the program.
  • the method of the present invention is to sample the single-stranded and double-stranded mosaic fluorescent light containing the target sequence of the polynucleotide of the sample.
  • the SYBR Green I reaction solution flows into the glass reaction chamber 1 together, and the temperature speed of the reaction chamber 1 is reduced to the appropriate annealing temperature Th.
  • the temperature range of Th is 4 ° C to 89 ° C, and then the reaction chamber is washed with a phosphate buffer solution. Excess sample polynucleotide chain and reaction solution in 1.
  • the temperature of the system in the reaction chamber 1 is slowly increased through the micro thermal cycler 8 with a heating rate of O.OI TV seconds, and the fluorescence intensity F of the gene chip 2 array is scanned with a scanner every 0.01 ° C, until the temperature rises to 100 ° C.
  • Th the annealing temperature
  • a high concentration of SYBR Green I dye will be embedded in the double strand. It will be excited by light at 470 ⁇ 490nm and emit 530nm by energy transfer.
  • the melting temperature Tm of the double strand ranges from 8 ⁇ to 100 ⁇ , and the double strand is melted, and the single strand of the sample polynucleotide target sequence will leave the corresponding probe on chip 2. Needle, SYBR Green I dye will also diffuse into the solution to reduce the fluorescence intensity velocity to zero. Melting point of double strand melting curve (F ⁇ T) ( Figure 2A) or derivative derivative melting curve (dF / dT ⁇ T) derived from continuous detection of fluorescence intensity by each probe on gene chip 2 ( Figure 2B The melting point Tm can be obtained at the peak center of).
  • F ⁇ T double strand melting curve
  • dF / dT ⁇ T derivative melting curve
  • F is the fluorescence intensity of the probe
  • T is the temperature
  • 1 is the melting curve when the polynucleotide target sequence is mismatched with the base of the probe
  • 2 is the base of the polynucleotide target sequence and the probe. Melting curve when perfectly matched.
  • dF / dT is the derivative of the fluorescence intensity of the probe with respect to temperature
  • T is the temperature
  • 3 is the melting curve when the polynucleotide target sequence is mismatched with the base of the probe
  • Tm is the peak center corresponding
  • the temperature value is the melting temperature.
  • Tmp is the temperature value corresponding to the center of the peak. It is both the melting temperature and the characteristic melting temperature of the probe. Tmp can be obtained using a polynucleotide target sequence standard that is perfectly matched to the probe. As long as the measured sample Tm is less than Tmp, it can be determined that the polynucleotide target sequence is mismatched with the base of the probe, because the binding force between the double strands is less than the perfect match between the polynucleotide target sequence and the base of the probe The binding force between double strands. Only when the measured sample Tm is equal to Tmp, can it be determined that the polynucleotide target sequence completely matches the base of the probe. Industrial applicability
  • the invention can be used for detecting the specific base sequence contained in the sample nucleotide:
  • the double-stranded nucleotide of the sample is heated to above 94 ° C, and the double-stranded strand is melted into single-stranded (the single-stranded nucleotide sample can also be obtained by other methods).
  • the single-stranded nucleotides are then flowed into the device of the present invention together with the reaction solution, and the temperature at each probe position on the gene chip is measured by a temperature scanning method.
  • the binding force between the double strands will decrease, and the corresponding melting temperature Tm will be less than Tmp. That is, the criterion for determining the correctness of the hybridization result of the chip array probe is that the melting temperature Tm is equal to the characteristic melting temperature Tmp of the probe. This can determine whether a single-stranded nucleotide in a sample contains a specific base sequence that is complementary to each probe sequence on the gene chip.
  • the invention can be used for the separation of nucleotides of different base sequences in the nucleotides of a sample: a single-stranded nucleotide is flowed into the device of the invention together with a reaction solution, and a probe containing only a single melting point temperature Tmp is selected first. Needle chip, using the temperature scanning method to monitor the melting temperature Tm between the single-stranded nucleotide of the sample and the probe at each probe position on the gene chip.

Description

四维参数枪测基因芯片上核酸杂交对的方法及装置 技术领域
本发明涉及一种基因检测技术，尤其是一种利用四维参数检测基因芯 片上核酸杂交对的方法及其装置 (G01N33/50)。 背景技术
在 1953年 Watson和 Crick等人提出了 DNA的双螺旋结构模型的概 念。他们指出：（l)DNA分子是由两条方向相反的平行多核苷酸链构成的， 这两条链在化学上具有相反方向。 （2)碱基成对有一定的规律： Chargaff 等应用层析法对多种生物 DNA的碱基组成进行分析， 发现 DNA中腺嘌 吟数目 (A)与胸腺嘧啶 (T)的数目相等， 胞嘧啶 (C)的数目与鸟嘌呤 (G)数目 相等。 因此， 在 DNA中有四种可能的碱基对： A-T、 T-A、 G-C、 C- G。
(3)两条链主要由碱基间的氢键相连： 碱基对的平面穿过螺旋轴， 约与螺 旋轴垂直。 AT间可形成两条氢键， GC 间可形成三条氢键。 同时， 对于 DNA双螺旋的结构稳定而言， 还需借助疏水键的作用力。 （4)由于四种碱 基对都适合此模型，每条链可以有任意的碱基顺序，但由于碱基成对的规 律性， 如一条链的碱基顺序已确定， 则另一条链必有相对应的碱基顺序。
由于 DNA双螺旋结构主要靠氢键和疏水键维系， 因此加热、 酸碱、 有机溶剂等凡是能破坏氢键和疏水键的因素都能引起变性， 使 DNA的双' 螺旋结构变为无规则线团。 不同变 ti DNA片段之间， 通过碱基互补配对 进行的 "复性 "称为杂交。 杂交不仅可以发生在 DNA与 DNA链间， 也 可在 DNA与 RNA链的同源序列之间进行。 杂交过程中两条互补的单链 DNA以非共价键方式形成双键杂合体。 当其中一条链的序列已知时， 通 过检测杂交过程，就可以探明未知 D N A样品中是否含有与已知序列互补 的 D N A存在。
基于上述原理已发展出了许多基因产品，其中基因传感器就是应用较 多的技术。
近年来，国内外有关基因传感器也称 D N A或核酸传感器的研究正成 试国生物传感器技术的研究热点， 基因传感器以其简易、快捷、价廉的独 特优越性，在分子生物学、医学检验和环境监测等领域具有广泛的应用前 景， 除基因序列分析、基因突变、基因检测和诊断外， 还涉及 D N A与药 物、 蛋白质分子间相互作用的研究等。
当前， 基因分析方法主要是在非均相体系中检测具体 D N A的序列， 比较常用的方法是核酸杂交法，核酸杂交是两条互补单链 D N A以非共价 键方式形成双键杂合体的过程，可以探明未知 D N A样品中是否含有与已 知序列互补的 D N A存在，最常用的方法是在某个固体支持物上固定一段 已知序列的基因， 然后利用其在溶液中同与之互补的寡核苷酸进行杂交， 从而实现液相中具体的 D N A检测。
近年来，人们对通过杂交方法来检测液相中具体 D N A序列的研究越 来越深入，通过杂交法检测 D N A序列有很重要的应用价值，主要应用在： 临床基因诊断、 法医学、 食品、 生物化学、 环境保护等领域， 而且基因的 检测方法也因生物素、地高辛、荧光染料等非放射性标记物的应用而变得 更加方便和安全， 特别是多聚酶链反应 PCR技术的应用， 使得基因检测 更加灵敏。
传统的 DNA杂交反应都要求使用标记方法来检测杂交信号， 这些方 法允许原位检测， 而且可以灵敏度很高。 如： PCR技术的检测限能达到 nmol/1; DNA计算机技术也提供了从大量混合体系中检测某一具体 DNA 序列的方法； 由于短波荧光和共聚焦显微镜技术的应用，荧光标记法已经 成为检测微量 DNA非常灵敏的常用方法。 目前现有的荧光标记法中， 基 因芯片（DNA芯片， 或基因微阵列）装置都只包括 XYZ三维参数， 例如 美国 Affymetrix公司生产的基因芯片 GeneCMp®。 这些基因芯片通常将 寡核苷酸 DNA探针固定在玻璃片基上， 寡核苷酸 DNA探针的碱基排列 组合和链的长度可以用 XYZ三维参数表征。 单链多核苷酸靶序列直接以 荧光染料标记，然后与芯片阵列探针杂交形成双股链，用扫描仪检测芯片 阵列荧光强度来获得样品信息。美国专利 US— 5,445,934和 US— 5,744,305 揭示了利用光刻合成技术制造的含大量 DNA检测位点的基因芯片装置， 这些装置中， 所有与固定在芯片上的寡核苷酸探针和其他 DNA探针序列 的杂交，都同时在同一温度进行。由于寡核苷酸探针的链长度和碱基组成 (GC含量） 不同， 其解链温度 （Tm)——即 50%的探针与靶核酸链分 离时的温度■ ~也不同， 因而最佳杂交温度必不相同。由于存在杂交温度 不一致的缺点， 所以结果的正确性无法保证， 也无法鉴别单碱基错配。为 了克服上述的局限性，美国专利 US— 6，238,868揭示了一种以电场为一自 由参数的微芯片杂交阵列， 加快了杂交的基本流程， 但是， 其制造复杂， 成本很高，样品多核苷酸靶序列要用荧光标记或者同荧光标记的另一个报 告探针相杂交，无法保证高通量全排列芯片结果的正确性， 限制了阵列杂 交的广泛应用。
目前，还有研究者开始利用非标记法来检测基因序列。研究较多的是 DNA生物传感器系统。 这些基因传感器主要分为三类： 一是光学基因传 感器，又分为荧光光纤基因传感器、表面增强拉曼基因探针和表面等离子 体共振基因传感器三类。 二是电化学基因传感器。 三是压电基因传感器。 但这些传感器的特异性和敏感度仍有待提供。 发明的公开
本发明的目的是为了提供一种四维参数检测基因芯片上核酸杂交对 的方法， 该方法是基于传统的利用 XYZ三维参数的检测核酸杂交对的基 础上，引入一温度作第四维参数来扫描样核酸杂交对的荧光标记物随温度 升高至解链温度时的荧光强度的变化而获得解链温度，通过与标准核酸杂 交对的解链温度的比较来判定杂交样品核苷酸单链的特征， 该方法简单、 准确、 灵敏度高、 特异性强。
本发明的另一目的在于提供一种四维参数检测基因芯片上核酸杂交对 的装置， 该装置的制造简单、 成本低， 即使基因的杂交温度不一致， 也能 保证高通量全排列芯片结果的正确性。
本发明的目的要通过如下措施来实现： ' 一种四维参数检测基因芯片上核酸杂交对的方法， 包括下述步骤：
(1)将基因芯片置于一可调温装置内；
(2) 将样品多核苷酸靶序列单链和含双股镶嵌式荧光染料反应液一 起流入容置有基因芯片的可调温装置内，将该调温装置内的温度降至退火 温度 Th， 使样品多核苷酸靶序列单链与基因芯片上的探针杂交形成核酸 双股链； 并由镶嵌在双股链的荧光染料标记； (3)然后升高装置内的温度， 其升温速度为 0.001-rC/秒， 且每升高 Δ Τ 用扫描仪扫描一次基因芯片阵列的荧光强度 F， 直至 100°C ; 其中 A T°C为升温速度中的每秒升温的温度； 当装置内温度升至核酸杂交双股 链的解链温度 Tm时，双股链解链，荧光染料扩散到溶液中使荧光强度,讯 速下降至零； 由扫描仪扫描基因芯片上的每个探针，连续检测荧光强度得 到双股链的熔解曲线的拐折点或衍生的导数熔解曲线，由上述曲线的峰中 心获得双股链的解链温度 Tm; 将该解链温度 Tm与已知配对正确的杂交 双股链的解链温度进行比较即可检测与基因芯片的探针的进行杂交的样 品多核苷酸序列单链的特征。
在上述 (2)还包括一用不含荧光染料的、 与退火温度同温的缓冲液洗 去装置内多余的样品多核苷酸链和反应液的步骤。
所述的退火温度 Th为 4-89°C。
所述的解链温度 Tm为 8-100 °C。
所述的升温速度为 o.oi-rc/秒。
所述的含双股镶嵌式荧光染料反应液选自 Molecular Probe公司生产 的 SYBR Green I反应液、 SYBR Green II反应液、 SYBR Gold中的一种。
本发明的另一目的可通过如下措施来实现：
一种四维参数检测基因芯片上核酸杂交对的装置， 包括可容置基因芯 片的容器、基因芯片、温控热循环装置、及设在容器上的液体进口和液体 出口；在可容置基因芯片的容器内放有基因芯片，容器与温控热循环装置 相连。
所述的温控热循环装置包括温度传感器、热循环装置和温控装置；其 中温度传感器与温控装置相连， 温控装置与热循环装置相连。
本发明的目的还在于提供一种四维参数检测基因芯片上核酸杂交对 的装置的应用。
一种四维参数检测基因芯片上核酸杂交对的装置用于 DNA样品的检 测和分离。
一种四维参数检测基因芯片上核酸杂交对的装置用于 RNA样品的检 测和分离。
本发明相比现有技术具有如下优点： 1、 本发明的方法是基于传统的利用 XYZ三维参数的检测核酸对基 础上，引入一温度作第四维参数来扫描核酸杂交对的荧光标记物随温度升 高至解链温度时的荧光强度的变化而获得解链温度，通过与标准核酸杂交 对的解链温度的比较来判定杂交样品核苷酸单链的特征，该方法简单、准 确、 灵敏度高、 特异性强。
2、 本发明引入了温度扫描方法， 解决了由于芯片阵列探针杂交温度 不一致性导致的结果是否正确的问题，对结果的阴阳判定给出了简单明确 的标准。
3、 本发明方法中的样品多核苷酸靶序列不用标记， 也无需使用另一 种荧光标记的报告探针， 方便了操作。
4、 本发明的装置结构简单， 可以与多种商品化芯片配套使用， 成本 较低；即使基因的杂交温度不一致，也能保证高通量全排列芯片结果的正 确性。 附图的简要说明
图 1是本发明装置的结构示意图。
图 2A是本发明的方法中的通过扫描基因芯片阵列探针点所得的熔解 曲线 （F~T)
图 2B是本发明的方法中的通过扫描基因芯片阵列探针点所得的导数 熔解曲线 （dF/dT~T) 实现本发明的最佳方式
参照图 1 , 为本发明的利用四维参数检测基因芯片上核酸杂交对的装 置， 该装置 9包括一个透明玻璃盒 1、 一块商品化的基因芯片 2、 一个温 控热循环装置 3和液体进口 5和一个液体出口 7。所述的温控热循环装置 3包括温度传感器 4、 热循环装置 8和温控装置 6; 所述的温控装置 6为 控温电脑；其中温度传感器 4与温控装置 6相连，温控装置 6与热循环装 置 8相连。控温电脑 6按照程序通过温度传感器 4和热循环装置 8来控制 玻璃盒 1内的反应液和基因芯片 2的温度。
本发明的方法是将样品多核苷酸靶序列单链和含双股镶嵌式荧光染 料 SYBR Green I反应液一起流入玻璃反应室 1， 将反应室 1的温度讯速 降低至适当退火温度 Th， Th的温度范围为 4°C〜89°C， 然后用磷酸缓冲 液洗去反应室 1内多余的样品多核苷酸链和反应液。通过微型热循环装置 8缓慢升高反应室 1内体系温度， 升温速度为 O.OI TV秒， 每升高 0.01 °C 用扫描仪扫描一次基因芯片 2阵列的荧光强度 F， 直到升温至 100°C。 只 要退火温度 Th足够低， 样品核酸单链就与芯片 2上探针形成双股链， 高 浓度的 SYBR Green I染料会镶嵌在双股链中，受 470〜490nm光激发经能 量传递会发出 530nm的特征荧光，当温度升至双股链的解链温度 Tm，解 链温度 Tm的范围为 8Ό~100Ό， 双股链解链， 样品多核苷酸靶序列单链 就会离开芯片 2上相应探针， SYBR Green I染料也会扩散到溶液中使荧 光强度讯速下降至零。由基因芯片 2上的每个探针连续检测荧光强度得到 的双股链的熔解曲线 （F〜T) (图 2A) 的曲折点或衍生的导数熔解曲线 (dF/dT~T) (图 2B)的峰中心可以获得解链温度 Tm。 图 2A中， F为探 针的荧光强度， T为温度， 1为多核苷酸靶序列与探针的碱基有错配对时 的熔解曲线， 2为多核苷酸靶序列与探针的碱基完全配对时的熔解曲线。 图 2B中， dF/dT为探针的荧光强度对温度求导数值， T为温度， 3为多核 苷酸靶序列与探针的碱基有错配对时的熔解曲线， Tm为峰中心对应的温 度值， 既解链温度， 4为多核苷酸靶序列与探针的碱基完全配对时的熔解 曲线， Tmp 为峰中心对应的温度值， 既解链温度， 也是探针的特征熔点 温度。 Tmp可以用与探针完全配对的多核苷酸靶序列标准品求得。 只要 实测的样品 Tm小于 Tmp， 就可以判定多核苷酸靶序列与探针的碱基有 错配对，因为其双链之间的结合力要小于多核苷酸靶序列与探针的碱基完 全配对时双链之间的结合力。 仅在实测的样品 Tm等于 Tmp时， 才可判 定多核苷酸靶序列与探针的碱基完全配对。 工业实用性
本发明可用于检测样品核苷酸中含有特定的碱基序列：
将样品核苷酸双链通过加热至 94°C以上， 将双链解链成单链 （也可 通过其它方法获得单链核苷酸样品）。 再将单链核苷酸与反应液一起流入 本发明的装置内，利用温度扫描方法测出在基因芯片上各个探针位置处的 样品单链核苷酸与探针之间的解链温度 Tm。如果探针与靶链的碱基完全 配对， 其双链间结合力最大，对应的解链温度 Tm也最大， 即等于探针的 特征熔点温度 Tmp。 如果靶链与探针之间有一个（或二个以上）碱基错 配， 其双链间的结合力将减小， 对应的解链温度 Tm将小于 Tmp。 即判 定芯片阵列探针的杂交结果正确性的标准是解链温度 Tm等于探针的特 征熔点温度 Tmp。 由此可以判定样品单链核苷酸中是否含有与基因芯片 上各个探针序列相互补的特定碱基序列。
本发明可用于对样品核苷酸中不同碱基序列核苷酸之间的分离： 将单链核苷酸与反应液一起流入本发明的装置，先选用只含特征熔点 温度 Tmp单一种的探针芯片， 利用温度扫描方法监测在基因芯片上各个 探针位置处的样品单链核苷酸与探针之间的解链温度 Tm。 将解链温度 Tm小于特征熔点温度 Tmp时冲洗出的液体放弃，解链温度 Tm等于或高 于特征熔点温度 Tmp时冲洗出的液体中只含有与探针序列相互补的特定 碱基序列核苷酸。

Claims

权利要求
1、一种四维参数检测基因芯片上核酸杂交对的方法，包括下述步骤：
(1)将基因芯片置于一可调温装置内；
(2) 将样品多核苷酸靶序列单链和含双股镶嵌式荧光染料反应液一 起流入容置有基因芯片的可调温装置内，将该调温装置内的温度降至退火 温度 Ήι， 使样品多核苷酸靶序列单链与基因芯片上的探针杂交形成核酸 双股链； 并由镶嵌在双股链的荧光染料标记； ' .
(3) 然后升高装置内的温度， 其升温速度为：0.001-rC/秒， 且每升高 A C用扫描仪扫描一次棊因芯片阵列的荧光强度 F， 直至 100°C ; 当装 置内温度升至核酸杂交双胶链的解链温度 Tm时， 双股链解链， 荧光染料 扩散到溶液中使荧光强度讯速下降至零；由扫描仪扫描基因芯片上的每个 探针，连续检测荧光强度得到双股链的熔解曲线的拐折点或衍生的导数熔 解曲线， 由上述曲线的峰中心获得双股链的解链温度 Tm; 将该解链温度 Tm与已知配对正确的杂交双股链的解链温度进行比较即可检测与基因 芯片的探针的进行杂交的样品多核苷酸序列单链的特征。
2、如权利要求 1所述的四维参数检测基因芯片上核酸杂交对的方法， 其特征在于在上述 (2)还包括一用不含荧光染料的、 与退火温度同温的缓 冲液洗去装置内多余的样品多核苷酸链和反应液的步骤。
3、如权利要求 1所述的四维参数检测基因芯片上核酸杂交对的方法， 其特征在于所述的退火温度 Th为 4-89°C。
4、如权利要求 1所述的四维参数检测基因芯片上核酸杂交对的方法， 其特征在于所述的解链温度 Tm为 8-100°C。
5、如权利要求 1所述的四维参数检测基因芯片上核酸杂交对的方法， 其特征在于所述的升温速度为 o.oi-rc/秒。
6、如权利要求 1所述的四维参数检测基因芯片上核酸杂交对的方法， 其特征在于所述的含双股镶嵌式荧光染料反应液选自 Molecular Probe 公 司生产的 SYBR Green I反应液、 SYBR Green II反应液、 SYBR Gold 中的一种。
7、 一种四维参数检测基因芯片上核酸杂交对的装置， 包括可容置基 因芯片的容器 (1)、 基因芯片 (2)、 温控热循环装置 (3)、 及设在容器上的液 体进口 (5)和液体出口 (7); 其特征在于在可容置基因芯片的容器 (1)内放有 基因芯片 (2)， 容器 (1)与温控热循环装置 (3)相连。
8、如权利要求 7所述的四维参数检测基因芯片上核酸杂交对的装置， 其特征在于所述的温控热循环装置 (3)包括温度传感器 (4)、 热循环装置 (8) 和温控装置 (6); 其中温度传感器 (4)与温控装置 (6)相连， 温控装置 (6)与热 循环装置 (8)相连。
9、 一种权利要求 7所述的四维参数检测基因芯片上核酸杂交对的装 置用于 DNA样品的检测和分离。
10、一种权利要求 7所述的四维参数检测基因芯片上核酸杂交对的装 置用于 RNA样品的检测和分离。
PCT/CN2002/000751 2002-10-24 2002-10-24 Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn WO2004038042A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CA002429101A CA2429101A1 (en) 2002-10-24 2002-10-24 Method and equipment to monitor nucleic acid hybridization on a dna chip using four-dimensional parameters
JP2004545671A JP2005519642A (ja) 2002-10-24 2002-10-24 ４次元パラメーターを用いたｄｎａチップ上での核酸ハイブリダイゼーションを検出するための方法及び装置
US10/380,112 US7101671B2 (en) 2002-10-24 2002-10-24 Method and equipment to monitor nucleic acid hybridization on a DNA chip using four-dimensional parameters
AU2002338167A AU2002338167A1 (en) 2002-10-24 2002-10-24 A 4-dimensional method and equipment of detecting nucleic acid hybrid pair on the gene chip
PCT/CN2002/000751 WO2004038042A1 (fr) 2002-10-24 2002-10-24 Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn
EP02772012A EP1437416A4 (en) 2002-10-24 2002-10-24 METHOD AND DEVICES FOR MONITORING NUCLEIC ACID HYBRIDIZATION ON A DNA CHIP USING FOUR-DIMENSIONAL PARAMETERS
CNB028022254A CN1164769C (zh) 2002-10-24 2002-10-24 四维参数检测基因芯片上核酸杂交对的方法及装置

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2002/000751 WO2004038042A1 (fr) 2002-10-24 2002-10-24 Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn

Publications (1)

Publication Number Publication Date
WO2004038042A1 true WO2004038042A1 (fr) 2004-05-06

Family

ID=32111535

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2002/000751 WO2004038042A1 (fr) 2002-10-24 2002-10-24 Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn

Country Status (7)

Country Link
US (1) US7101671B2 (zh)
EP (1) EP1437416A4 (zh)
JP (1) JP2005519642A (zh)
CN (1) CN1164769C (zh)
AU (1) AU2002338167A1 (zh)
CA (1) CA2429101A1 (zh)
WO (1) WO2004038042A1 (zh)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8367324B2 (en) * 2003-11-17 2013-02-05 Canon Kabushiki Kaisha Method for judging change in probe-bearing substrate, probe-bearing substrate and detecting apparatus
GB0419325D0 (en) * 2004-09-01 2004-09-29 Perkinelmer Ltd A method of analysing a sample including fluorescent labels and apparatus therefor
JP2007010413A (ja) * 2005-06-29 2007-01-18 Canon Inc 核酸ハイブリッドの融点測定方法及びそのための装置
JP2007054048A (ja) * 2005-07-27 2007-03-08 Sony Corp ハイブリダイゼーション検出方法
US11001881B2 (en) 2006-08-24 2021-05-11 California Institute Of Technology Methods for detecting analytes
US8048626B2 (en) 2006-07-28 2011-11-01 California Institute Of Technology Multiplex Q-PCR arrays
US11525156B2 (en) 2006-07-28 2022-12-13 California Institute Of Technology Multiplex Q-PCR arrays
US11560588B2 (en) 2006-08-24 2023-01-24 California Institute Of Technology Multiplex Q-PCR arrays
WO2008084672A1 (ja) * 2007-01-10 2008-07-17 Arkray, Inc. 光学検出装置の性能確認方法およびそれに用いる標準試薬
US7906316B2 (en) * 2007-07-05 2011-03-15 The Johns Hopkins University Apparatus for detecting molecules
DE102007055386B4 (de) 2007-11-20 2015-07-16 Boehringer Ingelheim Vetmedica Gmbh Verfahren zur Kalibrierung eines Sensorelements
EP2153901A1 (de) * 2008-08-01 2010-02-17 Eppendorf Ag Temperierungsvorrichtung mit Testmöglichkeit und Verfahren zum Testen einer Temperierungsvorrichtung
KR20100025328A (ko) 2008-08-27 2010-03-09 삼성전자주식회사 이중가닥 영역과 말단 단일가닥 영역을 포함하는 이중가닥 핵산 프로브가 고정된 마이크로어레이를 제조하는 방법
CN103635568B (zh) 2011-06-24 2016-04-27 株式会社日立高新技术 核酸扩增装置和核酸分析装置
US9708647B2 (en) 2015-03-23 2017-07-18 Insilixa, Inc. Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays
US9499861B1 (en) 2015-09-10 2016-11-22 Insilixa, Inc. Methods and systems for multiplex quantitative nucleic acid amplification
WO2017155858A1 (en) 2016-03-07 2017-09-14 Insilixa, Inc. Nucleic acid sequence identification using solid-phase cyclic single base extension
US10718758B2 (en) 2016-06-03 2020-07-21 International Business Machines Corporation Biosensor for optical detection of nucleotide sequence
US10373704B2 (en) 2016-06-03 2019-08-06 International Business Machines Corporation Reduction of surface nucleotide hybridization by optimizing a biosensor sensing surface area
US10712308B2 (en) 2016-06-03 2020-07-14 International Business Machines Corporation Biosensor for electrical detection of a nucleotide sequence
US11268134B2 (en) * 2017-09-29 2022-03-08 Boehringer Ingelheim Vetmedica Gmbh Sensor apparatus and method for testing a sample
EP3937780A4 (en) 2019-03-14 2022-12-07 InSilixa, Inc. METHODS AND SYSTEMS FOR TIMED FLUORESCENCE-BASED DETECTION

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1252453A (zh) * 1999-11-04 2000-05-10 徐社会 三维立体基因芯片及其制造方法
CN1284568A (zh) * 1999-08-13 2001-02-21 杨梦甦 利用白细胞分化抗原(cd)基因芯片的检测疾病的方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5143854A (en) 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5744101A (en) 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5681575A (en) 1992-05-19 1997-10-28 Westaim Technologies Inc. Anti-microbial coating for medical devices
US6114122A (en) * 1996-03-26 2000-09-05 Affymetrix, Inc. Fluidics station with a mounting system and method of using
GB9821989D0 (en) * 1998-10-08 1998-12-02 Hybaid Ltd Detection of nucleic acid polymorphism
CA2644688C (en) * 1999-04-20 2012-11-13 Kankyo Engineering Co., Ltd. Method for determining a concentration of target nucleic acid molecules, nucleic acid probes for the method, and method for analysing data obtained by the method
WO2001059144A1 (en) * 2000-02-10 2001-08-16 The Penn State Research Foundation Method of analyzing single nucleotide polymorphisms using melting curve and restriction endonuclease digestion
DE10027040A1 (de) * 2000-06-02 2001-12-06 Basf Lynx Bioscience Ag Verfahren zur Herstellung von DNA-Arrays

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1284568A (zh) * 1999-08-13 2001-02-21 杨梦甦 利用白细胞分化抗原(cd)基因芯片的检测疾病的方法
CN1252453A (zh) * 1999-11-04 2000-05-10 徐社会 三维立体基因芯片及其制造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1437416A4 *

Also Published As

Publication number Publication date
AU2002338167A1 (en) 2004-05-13
US7101671B2 (en) 2006-09-05
CA2429101A1 (en) 2004-04-24
JP2005519642A (ja) 2005-07-07
EP1437416A1 (en) 2004-07-14
US20040081974A1 (en) 2004-04-29
CN1164769C (zh) 2004-09-01
EP1437416A4 (en) 2004-07-14
CN1446264A (zh) 2003-10-01

Similar Documents

Publication Publication Date Title
WO2004038042A1 (fr) Methode quadridimensionnelle et appareil de detection d'une paire hybride d'acides nucleiques sur une puce a adn
JP4505776B2 (ja) 遺伝子検出システム、これを備えた遺伝子検出装置、検出方法、並びに遺伝子検出用チップ
Cheng et al. Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis
EP1297179A1 (en) Detection of nucleic acid hybridization by fluorescence polarization
AU2001261523A1 (en) Detection of nucleic acid hybridization by fluorescence polarization
EP2180066B1 (en) Single nucleotide polymorphism genotyping detection via the real-time invader assay microarray platform
US20140212874A1 (en) Enhanced probe binding
JP3752466B2 (ja) 遺伝子検査方法
JP2022037151A (ja) 対流流体装置における核酸の表面ベースの検出方法
US20100069253A1 (en) Impedance Spectroscopy Measurement of DNA
Ou et al. Rolling circle amplification-based biosensors
KR20120054882A (ko) 표적 핵산 검출 장치
WO2004050906A1 (fr) Procede de detection de la methylation de l'adn
KR20120045909A (ko) 복수 종의 표적 핵산을 실시간으로 검출하는 장치 및 방법
JP4320188B2 (ja) 一塩基置換検出方法及び一塩基置換検出用キット
US20100291695A1 (en) Detection of nucleic acid sequence modification
JP2002296280A (ja) キャピラリアレイを用いた遺伝子発現頻度解析方法
CN117051084A (zh) 一种利用无修饰探针的核酸检测方法及其应用
JP2020529865A (ja) 濃度に基づくｄｎａシーケンシングマシン
GB2360087A (en) Analytical method
Huang et al. Electrochemical real-time DNA amplification and detection on a microchip
Damrow et al. Optical, Electrochemical, and Magnetic DNA Biosensors–An Overview
WO1998054363A1 (en) Application of helix-random chain thermodynamics to nucleic acid analysis
JP2002000300A (ja) 簡易塩基配列確認法

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 028022254

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 10380112

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2002772012

Country of ref document: EP

Ref document number: 2429101

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2004545671

Country of ref document: JP

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWP Wipo information: published in national office

Ref document number: 2002772012

Country of ref document: EP