WO2004017062A1 - The method and device for accelerating molecular hybridization by ultrasonic wave - Google Patents

The method and device for accelerating molecular hybridization by ultrasonic wave Download PDF

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Publication number
WO2004017062A1
WO2004017062A1 PCT/CN2002/000882 CN0200882W WO2004017062A1 WO 2004017062 A1 WO2004017062 A1 WO 2004017062A1 CN 0200882 W CN0200882 W CN 0200882W WO 2004017062 A1 WO2004017062 A1 WO 2004017062A1
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Prior art keywords
hybridization reaction
ultrasonic
hybridization
liquid
reaction system
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English (en)
French (fr)
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WO2004017062A8 (en
Inventor
Gang Li
Wanli Xing
Dong Wang
Huafang Gao
Min Guo
Jing Cheng
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Tsinghua University
CapitalBio Corp
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Tsinghua University
Capital Biochip Corp
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Priority to AU2002354140A priority Critical patent/AU2002354140A1/en
Publication of WO2004017062A1 publication Critical patent/WO2004017062A1/zh
Publication of WO2004017062A8 publication Critical patent/WO2004017062A8/zh
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/80—Mixing by means of high-frequency vibrations above one kHz, e.g. ultrasonic vibrations
    • B01F31/85—Mixing by means of high-frequency vibrations above one kHz, e.g. ultrasonic vibrations with a vibrating element inside the receptacle
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00479—Means for mixing reactants or products in the reaction vessels
    • 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
    • C12Q1/6832—Enhancement of hybridisation reaction

Definitions

  • the present invention relates to a method for promoting a hybridization reaction and its hydrangea, and particularly to a method and a device for rapid and highly specific nucleic acid or nucleic acid binding protein detection.
  • the method and device can be applied to technical fields such as clinical diagnosis, basic research of life sciences, agriculture and environmental monitoring.
  • Hybridization is a basic process in molecular biology and an effective means for detecting specific nucleic acid and protein molecules in the fields of biology and medicine.
  • the conventional hybridization reaction method is to allow the target molecules and probe molecules participating in the reaction to mix freely (liquid-liquid system) or directly contact (liquid-solid system).
  • This method uses the free diffusion of molecules in a liquid medium to achieve contact between different components, thereby completing the reaction. Since free diffusion is a slower kinetic process, although this method is simple, the reaction takes a long time, the specificity is poor, and the uniformity is not good. Therefore, people have been searching for a method that can achieve rapid and highly specific hybridization reactions. Although there are currently some methods for accelerating the hybridization reaction, they all have their limitations.
  • the microelectronic gene chip designed by Nanogene in the United States uses the forward electrostatic force to attract the target molecules in the solution to the surroundings of the probe molecules to accelerate the hybridization reaction. After the hybridization reaction is completed, the reverse electrostatic force is loaded by changing the electrode polarity. The hybridized nucleic acid molecule is removed, thereby increasing the specificity of the hybridization reaction (see “Feng L., Nerenberg M. Electronic Microarray for DNA Analysis. Gen Ther. Mol. Biol., 1999, 4: 183-191").
  • this technology has the disadvantages of complicated processing and high cost.
  • the Lucidea full-automatic gene chip hybridization system produced by Amersham Biosciences of the United Kingdom accelerates the hybridization reaction by sucking the hybridization reaction liquid through mechanical components to promote mixing.
  • the device due to the moving parts of the device required by this technology, the device is relatively large and it is not easy to achieve miniaturization and integration.
  • the purpose of the present invention is to provide a method for promoting a hybridization reaction by using ultrasonic waves, so as to shorten the hybridization reaction time, improve the detection sensitivity, the specificity of the hybridization reaction, and the consistency of the hybridization signal; and simultaneously provide a simple structure and low cost. Simple operation, less time consuming, easy to realize automation, and can be used in hybridization reaction device of micro reaction system.
  • a method for promoting a hybridization reaction by using ultrasonic waves uses an ultrasonic generator to generate ultrasonic waves of a certain intensity, and puts a prepared solid-liquid hybridization reaction system or a liquid-liquid hybridization reaction system in an ultrasonic propagation path, and places the ultrasonic resonance element
  • the coupling medium between the hybridization reaction system and the ultrasonic wave is transmitted to the hybridization reaction system.
  • the high-frequency vibration and radiation pressure of the ultrasonic wave are used to form effective agitation and flow in the liquid medium of the hybridization reaction system.
  • the movement of the reaction molecules; and during the hybridization reaction, the power or frequency of ultrasonic radiation can be adjusted according to different hybridization reaction systems, and the perturbation intensity of the liquid medium can be changed, so as to promote the progress of the hybridization reaction.
  • the present invention also provides a device for implementing the above method.
  • the device includes a solid-liquid hybridization reaction system or a liquid-liquid hybridization reaction system containing two or more different components in a liquid phase, and a container equipped with the hybridization reaction system. It is characterized in that at least one ultrasonic resonance element is placed on the square, bottom or around the reaction system, and a coupling medium capable of transmitting sound waves is filled between the ultrasonic resonance element and the hybridization reaction system, and the ultrasonic resonance element is connected to the resonance circuit.
  • the intensity of liquid disturbance can be changed by adjusting the power of ultrasonic radiation to achieve the best reaction effect.
  • the specificity of the hybridization reaction can also be improved during the reaction by adjusting the power of ultrasonic radiation.
  • the binding force between the target molecule and the probe molecule is less than that of a complete pairing due to a certain mismatch of nucleic acid sequences of the same length.
  • FIG. 1 is a schematic structural diagram of a device according to an embodiment of the present invention.
  • FIG. 2 is a schematic structural diagram when a gene chip is placed on a resonance element for a hybridization reaction in the embodiment proposed by the present invention.
  • FIG. 3 is a front view of a piezoelectric ceramic plate as an ultrasonic resonance element in the embodiment proposed by the present invention View.
  • Fig. 4 is a rear view of a piezoelectric ceramic plate used as an ultrasonic resonance element in the embodiment proposed by the present invention.
  • Fig. 5 is a pseudo-color image of the fluorescence signal scanned by the hybridization results of the gene chip using conventional techniques.
  • FIG. 6 is a pseudo-color image of a fluorescent signal scanned by a hybridization result of the gene chip using the present invention.
  • FIG. 7 is a comparative analysis diagram of fluorescence signal intensity values of the hybridization result scan and the conventional hybridization result scan proposed in the embodiment of the present invention.
  • the device for promoting the hybridization reaction using ultrasonic waves of the present invention includes two parts: one is a so-called ultrasonic generator, and the other is a hybridization reaction system.
  • the hybridization reaction system may be a liquid-liquid reaction system or a liquid-solid reaction system, that is, one component is fixed on the surface or inside of a solid substrate, and the other component is in a liquid phase.
  • the material of the ultrasonic resonance element may not be limited to ceramic materials, and materials such as quartz may be used; the shape of the ultrasonic resonance element may be cylindrical, conical, cylindrical, cubic, etc., any shape that is favorable for generating ultrasonic resonance elements; the ultrasonic resonance element 1W ⁇ 200W ⁇ ⁇ ⁇ ⁇ ⁇ The volume can be changed between 10 cubic microns to 1000 cubic centimeters; the ultrasonic frequency of the ultrasonic resonance element can be changed between 20kH Z to 100MHz; the power of the ultrasonic generator can be changed between 0.1W to 200W.
  • the coupling medium filled between the ultrasonic resonance element and the hybridization reaction system can be any liquid or solid medium that can conduct sound waves.
  • the liquid medium can be water, glycerin, vegetable oil, etc .; the solid medium can be plastic, silicone rubber, or solidified ring. Oxygen resin and so on.
  • FIG. 1 is a specific embodiment of the present invention. This embodiment uses ultrasound to promote the hybridization reaction of a gene chip.
  • Piezoceramic sheet 2 with ring on the edge The shape of the rubber seal ring 4 is to form a groove to hold the coupling medium, so that the piezoelectric ceramic sheet 2 and the gene chip 6 placed on it maintain good contact, and ensure that ultrasound has a relatively good interface at the interface between different media.
  • High transmittance. 7 indicates the region on the gene chip where the nucleic acid probe dots are fixed.
  • the instrument is an optical detector based on laser-induced fluorescence detection.
  • the instrument can obtain the fluorescence intensity signal of each point on the gene chip in this example, and then obtain the corresponding hybridization sample concentration information.
  • the scanner parameter laser light source power was set to 80% of the instrument's maximum value
  • the detector photomultiplier tube (PMT) sensitivity was set to 65% of the instrument's maximum value
  • the scanning accuracy was set to 5 microns.
  • FIG. 3 and 4 are electrode arrangement diagrams of a piezoelectric ceramic sheet used in the ultrasonic resonance element in the embodiment.
  • Fig. 3 is a front view of the piezoelectric ceramic sheet
  • Fig. 4 is a back view of the piezoelectric ceramic sheet: 8 indicates the metal electrode plated on the surface of the ultrasonic piezoelectric ceramic sheet, 9 indicates the exposed piezoelectric material on the back of the ultrasonic piezoelectric ceramic sheet.
  • Figures 5 and 6 are pseudo-color diagrams of hybrid fluorescent signals. These two graphs compare a conventional hybridization result scan The scan and the scan results of ultrasound-assisted hybridization show the promotion effect of ultrasound on gene chip hybridization.
  • the hybridization system involved in this example is as follows: First, a series of capturer probes are fixed on the chip (aldehyde-modified glass slide), which are terminal amino-modified oligonucleotides, and the target molecule is longer than the probe. Some of the nucleotides, one of which is complementary to the probe, and the other of which is complementary to the fluorescently labeled reporter molecule added to the reaction system.
  • the hybridization solution contains the target molecule and the reporter molecule respectively.
  • the target molecule and the reporter molecule are pre-hybridized at 52 ° C, and then the probe on the chip Cross. Therefore, the amount of target molecules can be indirectly measured by the fluorescence intensity value of the fluorescently labeled molecules carried by the reporting molecules.
  • Capturer probe fixed to the chip surface (1) Capturer probe fixed to the chip surface:
  • the probe fixed to the dot matrix line 1 is 5 '— N3 ⁇ 4—T12— TCACAGACTGACCGAGG;
  • the probe fixed to the dot matrix line 2 is 5, -NH 2 -T12 -TCACAGACTGACCGAGT; the probe fixed to the dot matrix line 3 is 5 '-NH 2 -T12-TCACAGACTGACCGAGA;
  • FIG. 7 is a comparative analysis chart of fluorescence signal intensity values of the hybridization result scan and the conventional hybridization result scan proposed in the embodiment of the present invention. It can be seen from FIG. 7 that under the same other conditions, the application of ultrasound will increase the fluorescence signal intensity of the hybridization reaction by 4-5 times, thereby increasing the detection sensitivity. This shows that ultrasound has a certain promotion effect on hybridization. This experiment has been repeated 4 to 5 times, and all have obtained the same experimental results.
  • the present invention Compared with the traditional hybridization technology, the present invention has the following advantages and beneficial effects: it can significantly increase the intensity of the hybridization signal (the examples show that the signal intensity of the hybridization result assisted by ultrasound is 4 to 5 times higher than that of the conventional hybridization result signal), The hybridization reaction can reach the complete reaction state more quickly, and the reaction time can be shortened; the hybridization reactions at different positions on the surface of the solid substrate can be made more consistent, and the uniformity of the hybridization signal can be improved; the intensity of the false positive signal can be reduced, and the specificity of the hybridization reaction can be improved.
  • the device provided by the present invention has a simple structure and low cost.
  • the core components include only an ultrasonic resonance element, a corresponding resonance circuit, and an effective coupling medium. The operation is simple and easy to implement automation.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

一种利用超声波促进杂交反应的方法及装置 技术领域
本发明涉及一种促进杂交反应的方法及其華簟， 特别涉及快速及高特异 性核酸或核酸结合蛋白检测的方法及装置。 此方法及装置可应用于临床诊 断、 生命科学基础研究、 农业及环境监测等技术领域。
背景技术
杂交反应是分子生物学中的一个基本过程， 同时也是生物和医学等领域 中检测特定核酸和蛋白分子的有效手段。 目前常规的杂交反应处理方法是让 参与反应的靶标分子和探针分子自由混合（液液体系） 或直接接触（液固体 系） 。 这种方法是利用分子在液体介质中的自由扩散来实现不同组分之间的 接触， 从而完成反应的。 由于自由扩散是一个较慢的动力学过程， 所以这种 方法虽然简便， 但是反应耗时长， 特异性差， 均匀性不好。 因而人们一直在 寻求能够实现快速、 高特异性杂交反应的方法。 虽然目前也有一些手段用来 加速杂交反应， 但是都有很大的局限性。 比如美国 Nanogene 公司设计的微 电子基因芯片利用正向静电力将溶液中的靶标分子吸引到探针分子的周围从 而加速杂交反应， 杂交反应结束后再通过改变电极极性加载反向静电力将未 杂交的核酸分子去掉， 从而提高杂交反应的特异性 （参见 " Feng L. , Nerenberg M. Electronic Microarray for DNA Analysis. Gen Ther. Mol. Biol. , 1999, 4 : 183-191 " ) 。 但是此技术存在加工过程复杂， 费用 昂贵的缺点。 英国 Amersham Biosciences公司生产的 Lucidea全自动基因 芯片杂交系统则通过机械部件抽吸杂交反应液体促进混合来加速杂交反应。 但是由于此技术所需装置存在运动部件， 使得装置相对庞大， 不易实现微型 化和集成化。
发明公开
本发明的目的是提供一种利用超声波促进杂交反应的方法， 以达到缩短 杂交反应时间、 提高检测灵敏度和杂交反应的特异性以及杂交信号的一致 性； 并同时提供一种结构简单， 成本低廉， 操作简使， 耗时少， 易于实现自 动化， 并可用于微型反应系统的杂交反应装置。
本发明的目的是通过如下技术方案实现的： 一种利用超声波促进杂交反应的方法， 该方法利用超声波发生器产生一 定强度的超声， 将准备好的固液杂交反应体系或液液杂交反应体系置于超声 波传播途径中， 通过放置在超声波谐振元件与杂交反应体系之间的耦合介 质， 使超声波传导至杂交反应体系中； 利用超声波的高频振动及辐射压力在 杂交反应体系的液体介质中形成有效的搅动与流动， 通过液体的流动带动其 中的反应分子的运动； 并在进行杂交反应的过程中， 可根据不同的杂交反应 体系， 调节超声辐射功率或频率， 改变液体介质扰动强度， 从而实现促进杂 交反应的进行。
本发明还提供了一种实施上述方法的装置， 该装置包括固液杂交反应体 系或含有两种或多种不同组分的液相组成的液液杂交反应体系以及装有杂交 反应体系的容器， 其特征在于： 在反应体系的 ±方、 底部或四周至少放置一 个超声波谐振元件， 在超声波谐振元件与杂交反应体系之间填充可使声波传 导的耦合介质， 所述超声波谐振元件与谐振电路相连。
对于不同的杂交反应体系 （比如长短不同、 碱基组成不同的核酸杂交反 应体系） ， 由于存在反应动力学差异， 可以通过调节超声辐射功率， 改变液 体扰动强度， 使之达到最佳的反应效果。 同理， 利用靶标分子与不同探针分 子杂交反应时的反应动力学差异， 也可以在反应 程中通过调节超声辐射功 率来提高杂交反应的特异性。 比如对于基因芯片上的核酸杂交反应， 由于对 同一长度的核酸序列， 有一定错配情况时的靶标分子与探针分子的结合力要 小于完全配对情况。 如果超声辐射强度增加的结果使得液体对杂交液中核酸 分子的作用力大于有一定错配情况时的靶标分子与探针分子的结合力， 但又 小于完全配对情况时靶标分子与探针分子的结合力， 那么超声辐射的结果可 以保留完全配对而排斥错配， 从而提高基因芯片杂交的特异性。 附图说明
图 1为本发明提出的实施例装置结构示意图。
图 2为本发明提出的实施例中将基因芯片放置在谐振元件上进行杂交反 应时的结构示意图。
图 3为本发明提出的实施例中采用压电陶瓷片作为超声谐振元件的正面 视图。
图 4为本发明提出的实施例中采用压电陶瓷片作为超声谐振元件的背面 视图。
图 5为基因芯片采用常规技术杂交结果扫描荧光信号伪彩色图。
图 6为基因芯片采用本发明所进行的杂交结果扫描荧光信号伪彩色图。 图 7为本发明提出的实施例杂交结果扫描与常规杂交结果扫描的荧光信 号强度值对比分析图。
实施发明的最佳方式
下面结合附图进一步说明本 ¾：明的具体实施： 本发明利用超声波促进杂 交反应的装置包括两部分： 一部分即所谓超声波发生器， 另一部分即杂交反 应体系。 实际应用时， 杂交反应体系可以是液液反应体系， 也可以是液固反 应体系， 即一种组分被固定于固体基质表面或内部， 另一种组分在液相中。 超声波谐振元件的材料可不限于陶瓷材料， 可采用石英等材料； 超声波谐振 元件的形状可以是圆柱形、 锥形、 圆筒形、 立方形等任何有利于产生超声波 谐振的元件的形状； 超声波谐振元件的体积可以在 10立方微米到 1000立方 厘米之间变化； 超声波谐振元件所产生的超声波频率可以在 20kHZ 到 100MHz之间变化； 超声波发生器的功率可以在 0. 1W到 200W之间变化。 由 于不同的杂交反应体系存在反应动力学差异， 因此在进行具体操作时， 应 根据杂交反应体系的不同， 调节超声辐射功率或频率， 改变液体介质扰动强 度， 从而实现促进杂交反应的进行。 在超声波谐振元件与杂交反应体系之间 填充的耦合介质可以采用任何可使声波传导的液体或固体介质， 液体介质可 以是水、 甘油、 植物油等 ； 固体介质可以是塑料、 硅橡胶、 固化的环氧树 脂等。
图 1为本发明的一个具体实施例， 该实施例利用超声波促进基因芯片的 杂交反应。
( 1 ) 将一直径为 2. 5厘米、 频率为 1. 7丽 Z的圆形压电陶瓷片 2贴附于 一尺寸为 8. 5cmX 5cmX 2cm的塑料盒 1中， 压电陶瓷片 2通过塑料盒 1侧壁 所钻小孔引出导线 3与超声波谐振电路 5相接。 超声波谐振电路 5的输出功 率为 10W。 然后将导线引出孔用环氧树脂胶密封。 压电陶瓷片 2边缘套有环 形橡皮密封圈 4， 其作用是为了形成凹槽盛装耦合介质， 以使压电陶瓷片 2 与置于其上的基因芯片 6之间保持良好接触， 保证超声在不同介质之间界面 处有较高的透射率。 7指示基因芯片上固定有核酸探针点阵的区域。
(2 ) 将混合好的杂交液放到加热器上， 98°C下加热 4 分钟使双链结构 解链， 在 4°C下冷却 8分钟使单链结构得到保持， 52Ό下预杂交 15分钟， 4
°C下冷却 5分钟以上， 然后取出立即放入冰盒。
(3 ) 在压电陶瓷片 2上面环形橡皮密封圈 4形成的圆形凹槽内充满水 或甘油， 以利于超声波的传导， 然后将进行杂交反应的基因芯片 6 (以载玻 片为载体） 置于其上 （固定有探针的一面背对压电陶瓷片， 并使芯片 6上的 杂交点阵区域 7正好位于压电陶瓷片 2的正 ；方） 。
(4) 用移液枪在芯片 6的每个方阵上滴加 8 μ 1杂交液， 并迅速将装有 待杂交基因芯片的塑料盒 1上盖并以封口膜密封， 以防止水份的挥发丧失。 然后将塑料盒置于已调节恒温为 30 °C的杂交炉中， 同时打开超声波发生器 电源。 此超声波发生器周期性地开通和关闭， 每一循环的开通和关闭时间分 别为 2秒和 10秒。 在不同的杂交反应中， 超声波发生器可以采用连续发生 的方式或间断发生方式。
(5 ) 杂交反应进行 2小时后， 取出杂交盒， 快速取出芯片， 放到装有清 洗液 （0. 2% SDS) 的盒内， 然后置于摇床上振荡清洗 15分钟 （室温） ， 以去 离子水冲洗干净， 离心 2分钟（1500转 /分钟)甩干。
利用 Scanarray 4000 扫描仪扫描杂交芯片， 检测杂交结果。 仪器为一 基于激光诱导荧光检测的光学检测仪， 利用该仪器可获得本实例中基因芯片 上每个点的荧光强度信号， 进而获得对应的杂交样品浓度信息。 扫描仪的参 数激光光源功率设为仪器最大值的 80 %， 检测器光电倍增管 （PMT) 的灵敏 度设为仪器最大值的 65 %， 扫描精度设为 5微米。
图 3和图 4为实施例中的超声波谐振元件所釆用的压电陶瓷片的电极排 布图。 图 3为压电陶瓷片正面视图， 图 4为压电陶瓷片背面视图： 8指示超 声压电陶瓷片表面所镀金属电极， 9 指示超声压电陶瓷片背面裸露的压电材 料。
图 5和图 6为杂交荧光信号伪彩色图。 此二图比较了常规杂交结果扫描 图与超声辅助杂交结果扫描图， 显示了超声作用对基因芯片杂交反应的促进 效果。
本实例中涉及的杂交体系如下： 在芯片 （醛基修饰的玻片） 上先固定一 系列捕获子 （capturer) 探针， 它们为末端氨基修饰的寡核苷酸， 靶分子为 比探针长一些的核苷酸， 其中一段与探针互补， 它的另一段与反应体系中加 入的带有荧光标记的报告分子互补。 杂交液中分别含有靶分子与报告分子， 为提高芯片上的捕获子探针与靶分子的杂交效率， 先在 52°C下使靶分子与 报告分子预杂交， 然后再与芯片上的探针杂交。 由此靶分子的量可以通过报 告分子所带的荧光标记分子的荧光强度值间接测得。
从图中可以看出， 在施加超声的情况下， 杂交荧光信号可以得到明显增 强。
生物材料：
(1) 固定到芯片表面的捕获子 （capturer) 探针：
固定到点阵行 1上的探针为 5 ' — N¾—T12— TCACAGACTGACCGAGG;
固定到点阵行 2上的探针为 5， -NH2-T12 -TCACAGACTGACCGAGT； 固定到点阵行 3上的探针为 5 ' -NH2-T12-TCACAGACTGACCGAGA;
(2) 与靶分子杂交的报告分子 （reporter) ：
5' 一 GAACC TGGGG ACCCT GCGCG GCTAC TACTA CTAGTG —3,
(3) 靶分子：
HLA A区 2601型荧光不对称 PCR产物， 长度为 lkb。
试剂：
(1) 杂交液组成：
3.6μ 1 靶（target) DNA
1.0μ 1 报告分子 （reporter) (40ng/ul)
2.7μ 1 20XSSC
0.8μ 1 1%SDS
(2) 洗涤液组成：
2XSSC
0.1%SDS 图 7为本发明提出的实施例杂交结果扫描与常规杂交结果扫描的荧光信 号强度值对比分析图。 从图 7可以看出， 在其他条件相同的情况下， 施加超 声会使杂交反应的荧光信号强度增加 4-5倍， 从而增加检测的灵敏度。 说明 超声对杂交反应是有确定的促进作用的。 本实验已重复 4一 5 次， 均得到相 同的实验结果。
工业应用
本发明与传统杂交技术相比， 具有以下优点及有益效果： 可以显著提高 杂交信号的强度 （实施例显示经超声作用辅助的杂交结果信号强度比常规技 术杂交结果信号强度提高 4到 5倍） ， 使杂交反应更快地达到完全反应状态， 缩短反应时间； 可以使固体基质表面不同位置的杂交反应更趋一致， 提高杂 交信号的均匀性； 可以降低假阳性信号强度， 提高杂交反应的特异性。 另 外， 本发明提供的装置结构简单， 成本低廉， 核心元件仅有超声谐振元件和 相应谐振电路以及有效的耦合介质， 操作简便， 易于实现自动化 。

Claims

权利要求书
1.一种利用超声波促进杂交反应的方法， 其特征在于： 该方法利用超声 波发生器产生一定强度的超声， 将准备好的固液杂交反应体系或液液杂交反 应体系置于超声波传播途径中， 通过填充在超声波谐振元件与杂交反应体系 之间的耦合介质， 使超声波传导至杂交反应体系中； 利用超声波的高频振动 及辐射压力在杂交反应体系的液体介质中形成有效的搅动与流动， 通过液体 的流动带动其中的反应分子的运动； 并在进行杂交反应的过程中， 根据不同 的杂交反应体系， 调节超声辐射功率或频率， 改变液体介质扰动强度， 从而 实现促进杂交反应的进行。
2.按照权利要求 1所述的一种利用超声波促进杂交反应的方法， 其特征 在于所述超声波发生器的功率可在 0. 1W到 200W之间变化。
3.按照权利要求 1所述的一种利用超声波促进杂交反应的方法， 其特征 在于超声波发生器所产生的超声波频率可在 20kHz到 100MHz之间变化。
4.按照权利要求 1所述一种利用超声波促进杂交反应的方法， 其特征在 于： 所述的超声波发生器产生超声波的方式为连续进行或间断进行。
5.按照权利要求 1所述方法的装置， 该装置包括固液杂交反应体系或由 两种或多种不同组分的液相组成的液液杂交反应体系以及装有杂交反应体系 的容器， 其特征在于： 在反应体系的上方、 底部或四周至少放置一个超声波 谐振元件， 在超声波谐振元件与杂交反应体系之间填充可使声波传导的耦合 介质， 所述超声波谐振元件与谐振电路相连。
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JPH10108698A (ja) * 1996-10-02 1998-04-28 Toyobo Co Ltd 超音波を利用する核酸ハイブリダイゼーション反応方法およびそのための装置
JP2002062298A (ja) * 2000-08-22 2002-02-28 Mitsubishi Chemicals Corp ハイブリダイゼーション法及び生体試料検出法
CN1402967A (zh) * 2002-08-16 2003-03-19 清华大学 一种利用超声波促进杂交反应的方法及装置

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JPH10108698A (ja) * 1996-10-02 1998-04-28 Toyobo Co Ltd 超音波を利用する核酸ハイブリダイゼーション反応方法およびそのための装置
JP2002062298A (ja) * 2000-08-22 2002-02-28 Mitsubishi Chemicals Corp ハイブリダイゼーション法及び生体試料検出法
CN1402967A (zh) * 2002-08-16 2003-03-19 清华大学 一种利用超声波促进杂交反应的方法及装置

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