CN102559872B - Gene chip for detecting Angelman syndromes, using method and kit for gene chip - Google Patents

Gene chip for detecting Angelman syndromes, using method and kit for gene chip Download PDF

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CN102559872B
CN102559872B CN 201110410178 CN201110410178A CN102559872B CN 102559872 B CN102559872 B CN 102559872B CN 201110410178 CN201110410178 CN 201110410178 CN 201110410178 A CN201110410178 A CN 201110410178A CN 102559872 B CN102559872 B CN 102559872B
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gene chip
kit
detecting
gene
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CN102559872A (en )
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贺林
李笑天
秦胜营
郭奇桑
吴茜
龚小会
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上海交通大学
复旦大学附属妇产科医院
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Abstract

本发明涉及一种检测Angelman综合征的基因芯片及使用方法、试剂盒,所述基因芯片包括固相载体和固定在该固相载体上的探针,所述寡核苷酸探针为SEQ ID NO.1-50所示的核苷酸序列;所述使用方法包括如下步骤:(a)制备样品DNA片段;(b)荧光标记上述DNA片段;(c)洗脱上述标记产物;(d)将标记产物与所述基因芯片杂交;(e)扫描检测基因芯片的杂交信号,获得结果;本发明还涉及包含前述基因芯片的试剂盒。 The present invention relates to a method of detecting the use of gene chip and Angelman syndrome, a kit, the gene chip comprising a solid support and the probe immobilized on the solid support, the oligonucleotide probe is SEQ ID the nucleotide sequence shown NO.1-50; said method comprising the steps of using: a sample DNA fragment (a) preparation; (b) a fluorescent labeled DNA fragment described above; (c) eluting the labeled product; (d) the product of the marker gene chip hybridization; (e) gene chip hybridization scan test signal, the result is obtained; the present invention further relates to a gene chip comprising the kit. 本发明的基因芯片操作步骤简单,检测特异性高,稳定性好,从样本抽提到得到扫描结果可在一个工作日内完成,成本低,适用于临床患者基因突变检测、产前诊断和正常人杂合子携带者检测。 GeneChip Operating steps of the present invention is simple, high detection specificity, stability, obtained from the sample extracted into scan results can be completed in one working day, low cost, suitable for clinical gene mutation in patients, and normal prenatal diagnosis person heterozygous carriers detected.

Description

检测Angelman综合征的基因芯片及使用方法、试剂盒 Gene chip and Angelman syndrome using a detection kit

技术领域[0001] 本发明涉及一种生物技术领域的基因芯片及使用方法、试剂盒,具体涉及一种检测Angelman综合征的基因芯片及使用方法、试剂盒。 Technical Field [0001] The present invention relates to the field of biotechnology and gene chips using the methods, kits, particularly relates to a method for detecting gene chip method and the use of the kit Angelman syndrome.

背景技术 Background technique

[0002] Angelman综合征(Angelman syndrome, AS),又名快乐木偶综合征,是一种基因组印记性疾病,最早由Hurry Angelman在1965年报道。 [0002] Angelman syndrome (Angelman syndrome, AS), also known as happy puppet syndrome, is a genomic imprinting diseases, first reported by the Hurry Angelman in 1965. 该病在临床上表现为复杂的多系统异常,主要I临床症状包括小头畸形、快乐木偶样的共济失调步态、癫痫、EEG异常、过度兴奋和发作性大笑等。 In the clinical manifestations of the disease complex, multi-system abnormalities, mainly I clinical symptoms include microcephaly, happy puppet-like gait ataxia, epilepsy, EEG abnormalities, excessive excitement and episodes of laughing and so on. 母源15号染色体qll-13区域的异常是导致该病发生的主要原因,目前已明确有4种分子病理机制会导致AS的发生。 Maternal chromosome 15 abnormalities qll-13 area is the main cause of the occurrence of the disease, has clearly there are four kinds of pathological molecular mechanisms lead to the occurrence of AS. 国外报道AS的发病率约为 Foreign reports about the incidence of AS

2.5/100000-10/100000。 2.5 / 100000-10 / 100,000. 目前的研究表明4种明确的机制会导致Angleman综合征的发生:(I)母源染色体15qll.2-13关键区域发生4~6Mb的大片段缺失(占患儿的70% ),UBE3A基因位于这一区域痰,因此母源UBE3A基因表达缺失;(2) 15号染色体父源单亲二体性(uniparental disomy, UPD, 5% ),即患儿的两条15号染色体都来自父源,缺乏母源15号染色体,因此缺乏母源的UBE3A基因,而父源UBE3A基因不具有表达活性;(3)印记缺损(imprinting defect, ID, 5% ),母源15qll.2_13区域呈现异常的印记状态,从而引起UBE3A基因表达出现异常;(4)母源UBE3A基因发生突变(10%),无法正常表达。 Current research shows that four kinds of clear mechanisms can lead to Angleman syndrome occurs: (I) the maternal chromosome 15qll.2-13 key areas a major deletions 4 ~ 6Mb of (70% of children), UBE3A gene is located sputum this region, so UBE3A maternal gene deletion; (2) the paternal chromosome 15 uniparental disomy (uniparental disomy, UPD, 5%), i.e., No. 15 two children chromosomes from the parent source, the lack of maternal chromosome 15, thus lacking maternal UBE3A gene, while having no paternal gene expression UBE3A activity; (3) imprinting defect (imprinting defect, ID, 5%), maternal 15qll.2_13 region exhibiting the abnormal state of the mark , thereby causing the abnormal gene expression appears UBE3A; (4) maternal UBE3A mutated genes (10%), it can not be expressed properly. 这4种机制都会间接或直接引起UBE3A基因的低表达或不表达,从而使患者产生AS特征性的临床表现。 The four mechanisms are directly or indirectly caused UBE3A low or no expression of the gene, so that patients have clinical features of each AS. 另有约10%的AS患者其致病机制尚未明确。 While about 10% of AS patients whose pathogenesis is not yet clear.

[0003] 基于AS的发病机制不同,已建立了多种实验室诊断方法,其中MS-PCR是最主要和最常用的诊断方法。 [0003] Based on the different AS pathogenesis, we have established a variety of laboratory diagnostic methods, wherein the MS-PCR is the most important and most commonly used diagnostic method. 由于在AS的分子机制中,15qll_13缺失、父源单亲二倍体和印记缺陷均表现为甲基化状态的异常,涉及这3种机制的病例均可应用MS-PCR进行诊断,并且这3种机制的病例共占AS的80%,因此可首先应用MS-PCR方法对病例进行初步诊断。 Because the molecular mechanisms of AS, 15qll_13 deletion, uniparental disomy and paternal imprinting defect showed an abnormal methylation status, cases involving these three mechanisms of MS-PCR can be applied for diagnosis, and these three mechanism cases accounted for 80% AS, and thus may be first applied to cases of MS-PCR method of initial diagnosis. 此外,还可以使用FISH的方法,检出率为70%。 In addition, the FISH method may also be used, the detection rate of 70%. 然而尚无一种快速、低成本、大通量和自动化检测Angelman综合征基因突变的方法。 However, there is no a fast, low-cost, high-throughput and automated method for detection of gene mutation Angelman syndrome.

[0004] 比较基因组杂交(CGH)芯片分析技术是一种突破性技术,能够支持研究人员通过微阵列准确研究与疾病有关的染色体的变化。 [0004] comparative genomic hybridization (CGH) chip analysis technique is a breakthrough technology to support researchers precise changes associated with the disease chromosome by microarray. 比较基因组杂交(CGH)芯片分析的原理是在一张芯片上用两份标记不同荧光素的样品(检测样品和对照样品)同时进行杂交,从而快速而又直观地检测实验样品和对照样品之间基因组DNA的拷贝数量的差异。 Microarray analysis principle comparative genomic hybridization (CGH) is a chip on two samples labeled with different fluorescent pigment (test sample and control sample) hybridized simultaneously to quickly and intuitively detected between the experimental and control samples differences in the number of copies of genomic DNA. CGH应用于疾病遗传学的研究,可以提供一个全基因组的扫描图,提示样本DNA在整个染色体组的哪个特定位置存在扩增或缺失,那些发生扩增的区域,极有可能存在致病基因,而缺失区域即可能包含一些抑制基因。 CGH used to study the genetics of the disease, may provide a scan of the whole genome, suggesting the presence of sample DNA amplification or deletion of specific sites in the whole genome, those regions of the amplification occurs, there is most likely causative gene, i.e., the region may contain deletions suppressor gene. 因此,需要一种新的技术方案来克服现有技术的上述缺陷和不足。 Therefore, a new technical solution to overcome the above drawbacks and deficiencies of the prior art.

发明内容 SUMMARY

[0005] 本发明的目的在于克服现有技术的不足,提供一种检测Angelman综合征的基因芯片及使用方法、试剂盒。 [0005] The object of the present invention is to overcome the disadvantages of the prior art, and to provide the use of gene chip method, kits for detecting Angelman Syndrome. 本发明的基因芯片操作步骤简单,检测特异性高,稳定性好,时间短,成本低。 GeneChip Operating steps of the present invention is simple, high detection specificity, stability, short time, low cost. 本发明还提供了一种检测Angelman综合征的基因芯片的使用方法及试剂盒。 The present invention further provides a method of using a gene chip for detecting kits and Angelman Syndrome.

[0006] 本发明的目的是通过以下技术方案实现: [0006] The object of the present invention is achieved by the following technical solution:

[0007] 第一方面,本发明提供一种检测Angelman综合征的基因芯片,包括固相载体和固定在该固相载体上的寡核苷酸探针,所述寡核苷酸探针为SEQ ID N0.1~50所示的核苷酸序列。 [0007] In a first aspect, the present invention provides a gene chip for detecting Angelman syndrome, comprising the oligonucleotide probe and solid support immobilized on the solid support, the oligonucleotide probe is SEQ the nucleotide sequence shown N0.1 ~ 50 ID.

[0008] 优选地,所述固相载体选自载玻片、硅片、硝酸纤维素膜、尼龙膜或聚苯乙烯。 [0008] Preferably, the solid support is selected from glass slides, silicon, nitrocellulose membrane, nylon membrane or polystyrene.

[0009] 第二方面,本发明还涉及前述检测检测Angelman综合征的基因芯片的使用方法,包括以下步骤: [0009] In a second aspect, the present invention also relates to the use of gene chips Angelman syndrome detected by the detector, comprising the steps of:

[0010] (a)制备样品DNA片段; [0010] (a) Preparation of sample DNA fragments;

[0011 ] (b)荧光标记上述DNA片段; [0011] (b) the above-described fluorescent-labeled DNA fragments;

[0012] (c)洗脱上述标记产物; [0012] (c) eluting the labeled product;

[0013] (d)将标记产物与所述基因芯片杂交; [0013] (d) The product of the marker gene chip hybridization;

[0014] (e)扫描检测基因芯片的杂交信号,获得结果。 [0014] (e) a scanning signal detecting gene chip hybridization results were obtained.

[0015] 第三方面,本发明还涉及一种用于检测Angelman综合征的试剂盒,所述试剂盒包括权利要求1所述的基因芯片。 [0015] In a third aspect, the present invention relates to a detection kit for Angelman syndrome, said kit comprising a microarray according to claim 1.

[0016] 优选地,所述试剂盒还包括标记物,所述标记物为Cy3_dUTP。 [0016] Preferably, the kit further comprises a marker, the marker is Cy3_dUTP.

[0017] 优选地,该试剂盒还包括标记物,所述标记物为Cy5_dUTP。 [0017] Preferably, the kit further comprises a marker, the marker is Cy5_dUTP.

[0018] 本发明具有如下的有益效果:本发明的基因芯片操作步骤简单,检测特异性高,稳定性好,该芯片多次试验重复性高;时间短,从样本抽提到得到扫描结果可在一天内完成。 [0018] The present invention has the following beneficial effects: GeneChip Operating steps of the invention is simple, high detection specificity, stability, repeatability test multiple chip; time is short, the scan obtained from the sample results may be extracted into the completed in one day.

具体实施方式 detailed description

[0019] 下面结合具体实施例,进一步阐述本发明。 [0019] The following embodiments with reference to specific embodiments, further illustrate the present invention. 应理解为:这些实施例仅用于说明本发明而不用于限制本发明的范围。 It should be understood as follows: These examples serve only to illustrate the present invention and are not intended to limit the scope of the invention. 下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等分子克隆:实验室手册见New York Cold Spring HarborLaboratory出版社1989年版中所述的条件,或按照制造厂商所建议的条件。 Experimental methods without specific conditions in the examples below, are performed under routine conditions, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual Cold York see conditions the Spring Harbor Laboratory Press, New Edition, 1989 was used, or as recommended by the manufacturer condition.

[0020] 实施例 [0020] Example

[0021] 本实施例的基因芯片由固相载体和固定在该固相载体上的寡核苷酸探针,所述寡核苷酸探针为SEQ ID N0.1~50所示的核苷酸序列,所述固相载体为玻片。 [0021] The gene chip according to the present embodiment is composed of an oligonucleotide probe to a solid support and immobilized on the solid support, the oligonucleotide probe nucleosides shown as SEQ ID N0.1 ~ 50 acid sequence, the solid support is a slide. 本实施例基因芯片的固相载也可选用硝酸纤维素膜、硅片、尼龙膜或聚本乙烯。 Microarray solid-phase carrier of the present embodiment may also choose embodiment nitrocellulose, silicon, nylon or polyethylene film of the present ethylene. 本实施例的寡核苷酸探针由安捷伦公司生产,同时本实施例的基因芯片也由安捷伦公司按照常规方法生产。 Oligonucleotide probe according to the present embodiment is manufactured by Agilent Technologies, and gene chip according to the present embodiment is also manufactured by Agilent according to conventional methods.

[0022] 本实施例基因芯片的使用方法,步骤如下: [0022] using the method of the present embodiment microarray embodiment, the following steps:

[0023] (I)样品DNA片段的制备 Preparation (I) of sample DNA fragments [0023]

[0024] 取患者、正常人血液,采用常规方法抽提全基因组DNA(gDNA)。 [0024] Patients taking normal human blood, the extraction with a conventional genomic DNA (gDNA).

[0025] 将水浴锅温度调到37°C和65°C,溶化10 XBuffer C (见下表),短暂涡旋震荡后离心数秒。 [0025] The water bath temperature to 37 ° C and 65 ° C, melt 10 XBuffer C (see table below), after vortexing briefly centrifuged for a few seconds.

[0026] 对每个反应来说,将gDNA加入到适当的无核酸酶的PCR管中,并加入无核酸酶的水使反应体积达到10.1 μ L. [0026] For each reaction, the gDNA was added to the appropriate nuclease-free PCR tube and added nuclease-free water and the reaction volume to 10.1 μ L.

[0027] 在冰上准备酶切混合母液,按顺序加入以下试剂: [0027] Preparation digestion master mix on ice, the following reagents were added sequentially:

Figure CN102559872BD00051

[0029]其中,IOXBuffer C 由Rsa I 提供;Acetylated BSA 由Alu I 提供;Promega AluI 的货号为Promega p/n R6281 ;Promega Rsa I 的货号为Promega p/n R6371。 [0029] wherein, IOXBuffer C by Rsa I; Acetylated BSA by Alu I; Promega AluI item number Promega p / n R6281; Num Promega Rsa I of Promega p / n R6371.

[0030] 将2.9 μ L酶切反应液加入到gDNA反应管中,使之体积达到13 μ L。 [0030] The enzyme 2.9 μ L was added to the reaction gDNA reaction tube, so as to reach the volume of 13 μ L. 上下颠倒使之混匀。 Upside down so that mix. 进行PCR反应,反应程序为:37°C运行2小时,65°C运行20分钟。 The PCR reaction, the reaction procedure was: 37 ° C Run 2 hours, 65 ° C Run 20 minutes.

[0031] 反应后将反应管转移到冰上,取2 μ L酶切好的gDNA,配置0.8%琼脂糖胶,用SYBRGold染色,跑电泳检测酶切是否完全。 After the reaction [0031] The reaction tube was transferred onto ice, 2 μ L taken good digestion of gDNA, configured 0.8% agarose gel, stained with SYBRGold, digested electrophoresis run is complete. 大部分酶切产物长度应在200bp-500bp。 Most of the length of the cleavage product should be 200bp-500bp.

[0032] (2) DNA的荧光标记 Fluorescent label [0032] (2) DNA of

[0033]在每个反应管中加入 2.5 μ L 随机引物(Agilent Genomic DNA Labeling Kit,Agilent p/n 5188-5309),使之体积达到13.5 μ L,轻轻上下颠倒进行混匀。 [0033] Add 2.5 μ L random primer (Agilent Genomic DNA Labeling Kit, Agilent p / n 5188-5309) in each reaction tube, so that the volume to 13.5 μ L, mix gently for upside down. 进行PCR反应,反应程序为:95°C变性3分钟。 The PCR reaction, the reaction procedure was: 95 ° C denaturation for 3 minutes. 将反应管转移至冰上5min。 The reaction tube was transferred to ice 5min.

[0034] 准备标记混合母液,按顺序加入以下试剂: [0034] Preparation marker master mix, the following reagents were added sequentially:

Figure CN102559872BD00052

[0036] #Agilent Genomic DNA Labeling Kit, Agilent p/n 5188-5309。 [0036] #Agilent Genomic DNA Labeling Kit, Agilent p / n 5188-5309.

[0037] 将11.5 μ L标记反应液加入到gDNA反应管中,使之体积达到25 μ L。 [0037] The labeling reaction was 11.5 μ L gDNA was added to the reaction tube, so as to reach the volume of 25 μ L. 轻轻上下颠倒使之混匀。 Mix gently upside down to make it. 进行PCR反应,反应程序为:37°C运行2小时,65°C运行10分钟。 The PCR reaction, the reaction procedure was: 37 ° C Run 2 hours, 65 ° C run for 10 minutes. 将反应管转移到冰上。 The reaction tube was transferred onto ice. 其中Cy3-dUTP标记正常人gDNA,Cy5_dUTP标记患者gDNA。 Wherein Cy3-dUTP labeled normal human gDNA, Cy5_dUTP numerals patient gDNA.

[0038] (3)标记gDNA的洗脱 Elution [0038] (3) a labeled gDNA

[0039] 加入430yL的IX TE (pH 8.0)到每个反应管中。 [0039] Add IX TE (pH 8.0) 430yL to each reaction tube. 将一个Microcon YM-30过滤柱(Millipore p/n 42410)放入一个1.5mL离心管中,并且将标记好的gDNA加到滤膜上。 A filtration column Microcon YM-30 (Millipore p / n 42410) placed in a 1.5mL centrifuge tube, and applied to the membrane The labeled gDNA. 室温下8000g离心lOmin。 8000g centrifugation at room temperature for lOmin. 弃滤液。 The filtrate was abandoned. 加入480 μ L的1ΧΤΕ(ρΗ8.0)到每个过滤柱中。 Was added 480 μ L of 1ΧΤΕ (ρΗ8.0) to each filter column. 室温下8000g离心lOmin。 8000g centrifugation at room temperature for lOmin. 弃滤液。 The filtrate was abandoned.

[0040] 倒置过滤柱,放入一个新的1.5mL离心管中。 [0040] Invert filtration column into a new 1.5mL centrifuge tube. 室温下8000g离心Imin收集纯化样本。 Imin 8000g centrifugation to collect the purified sample at room temperature. 检查并记录每管洗出液的体积。 Check and record each tube eluate volume. 如果体积超出10 μ L,将洗出液重新加到滤膜上,室温下8000g离心lmin。 When the volume exceeds 10 μ L, was again added to the eluate on a filter, 8000g centrifugation lmin at room temperature. 弃滤液。 The filtrate was abandoned.

[0041] 重复上述两步直到样本体积均小于等于10 μ L。 [0041] Repeat the above two steps until the sample volume is less than equal to 10 μ L. 加入IX TE (pH 8.0)使最终体积达到10 μ L。 Was added IX TE (pH 8.0) to achieve a final volume of 10 μ L. 从每个样本中取1.5 μ L检测产量和比活,使用NanoDrop ND-1000UV-VIS分光光度计(由Thermo Fisher Scientific Inc 提供)测量。 Taken from each sample and 1.5 μ L Yield detecting specific activity using NanoDrop ND-1000UV-VIS spectrophotometer (supplied by Thermo Fisher Scientific Inc) measurement.

[0042] 将等量的分别用Cy5和Cy3标记的两种样本在一个新的1.5mL耐高温的离心管中混合,达到最终体积158 μ Lo [0042] Equal amounts of two kinds of samples were used Cy5 and Cy3-labeled mixed in a new refractory 1.5mL centrifuge tube, to a final volume of 158 μ Lo

[0043] (4)样品的预处理 [0043] Pretreatment (4) of the sample

[0044] 向装有IOXBlocking Agent冻干粉的小瓶中加入1350 μ L无核酶的水。 [0044] L 1350 μ no ribozyme is added to the water containing vial of lyophilized powder IOXBlocking Agent. 在使用或保存之前,室温放置60min,涡旋振荡混匀。 Before use or storage, placing 60min at room temperature, vortex mix.

[0045] 将水浴锅调至95°C和37 °C。 [0045] The water bath was adjusted to 95 ° C and 37 ° C.

[0046] 在一个无核酶的离心管中按顺序加入以下试剂: [0046] In one non-ribozyme sequence centrifuge tube the following reagents were added:

[0047] [0047]

Figure CN102559872BD00061

[0048]其中,Cot-1DNA 的货号为Invitrogen p/n 15279-011 ; [0048] wherein, Cot-1DNA item number Invitrogen p / n 15279-011;

[0049] Agilent IOXBlocking Agent 与Agilent 2XHybridization Buffer 的货号为Agilent p/n 5188-5220。 [0049] Agilent IOXBlocking Agent with Agilent 2XHybridization Buffer item number Agilent p / n 5188-5220.

[0050] 上下颠倒混合样品,随后短暂离心。 [0050] The sample was mixed upside down, and then briefly centrifuged. 将样品管转移到95°C水浴锅中水浴保温3min。 The sample tube was transferred to a water bath at 95 ° C water bath for incubation 3min. 立即将样品管转移到37°C水浴锅中水浴保温30min。 Samples were immediately transferred into tubes 37 ° C water bath water bath for 30min. 将样品管拿出水浴锅,17900g离心I分钟,收集管底样品。 The sample tube out water bath, 17900g I minute centrifugation, the sample collection tube bottom.

[0051] (5)芯片杂交 [0051] (5) microarray hybridization

[0052] 将一张干净的垫片放在杂交室(Agilent p/n G2534A)底部,使垫片标志朝上,并与杂交室底部的长方形区域对齐。 [0052] The pad is placed a clean hybridization chamber (Agilent p / n G2534A) at the bottom, the spacer logo facing rectangular region and aligned with the bottom of the hybridization chamber. 确保垫片与杂交室底部同高。 Make sure the gasket and the bottom of the hybridization chamber with high.

[0053] 缓慢地以画圈的方式将40μ L杂交样品混合液加到垫片上的垫圈内区域。 [0053] slowly in a circular motion to 40μ L hybridization mixture was added to the sample in the spacer region of the gasket. 将芯片的活性面朝下放在垫片上,使得印有数字条形码的一面朝上,而印有“Agilent”标志的条形码一面朝下。 The active face is placed on the chip pad, so that the digital barcode printed side up, and printed with "Agilent" barcode mark side down. 确保两者相互对齐。 Make sure both are aligned with each other. 将杂交室盖子盖在“垫片-杂交样品-芯片”三明治结构上,滑动夹具使之固定。 The hybridization chamber lid on the "spacer - - sample chip hybridization" sandwich, the slide clamp to fix it. 手动加固杂交室上的夹具。 Hybridization chamber manual jig reinforcement. 垂直旋转组装好的杂交室以润湿芯片,观察气泡是否可以自由流动。 Rotating the assembled vertical chamber to wet hybridization chip, to observe whether bubbles can flow freely. 如果发现静止的气泡,可以将杂交室放在桌面上通过轻轻敲打芯片或垫片使之移动。 If it is found bubble stationary hybridization chamber may be placed gently tap the chip pad to move it through or on the desktop. 将组装好的杂交室固定在旋转器上,放入65°C的杂交炉中以20rpm的转速转动,杂交24小时。 The assembled hybridization chamber is fixed on a rotator, into a 65 ° C hybridization oven at the rotation speed of 20rpm, hybridized for 24 hours. [0054] (6)芯片洗脱和扫描 [0054] (6) scanning and eluted chips

[0055]在室温下将染色缸 1#装满Wash Buffer I (Agilent Oligo aCGH Wash Buffer 1,Agilent p/n 5188-5221)。 [0055] at room temperature staining jar filled with # 1 Wash Buffer I (Agilent Oligo aCGH Wash Buffer 1, Agilent p / n 5188-5221). 将一个芯片架放入染色缸2#。 The chip tray in a dyeing cylinder # 2. 并往染色缸2#中放入一个磁力搅拌子。 To Coplin jars and placed in a # 2 magnetic stirrer. 室温下往染色缸2#中装入足够多的Wash Buffer I使之足以覆盖芯片架。 Coplin jars at room temperature for 2 to # charged enough Wash Buffer I to be sufficient to cover the chip carrier. 将染色缸2#放在磁力搅拌器上。 The dyeing cylinder # 2 on a magnetic stirrer. 将在37°C烘箱中预热过的染色缸3#放在带有加热元件的磁力揽拌器上,装入3/4 体积的预热过的Wash Buffer 2 (Agilent Oligo aCGH Wash Buffer2,Agilent p/n 5188-5222),再放入一个磁力搅拌子。 The preheated oven at 37 ° C on a staining jar # 3 with a heating element placed on a magnetic stirrer embrace charged with 3/4 volume of preheated Wash Buffer 2 (Agilent Oligo aCGH Wash Buffer2, Agilent p / n 5188-5222), and then placed in a magnetic stirrer. 打开加热元件,保持Wash Buffer2的温度于37°C;用温度计控制温度。 Turn on the heating element, maintaining the temperature in Wash Buffer2 37 ° C; with a thermometer to control the temperature. 将一个杂交室从杂交炉中拿出来并计时。 A hybridization chamber and timed out from the hybridization oven. 记录杂交过程中是否形成气泡以及所有气泡是否旋转自由。 Recording the formation of bubbles during hybridization if any air bubbles and is rotating freely. 拆卸杂交室。 Removing hybridization chamber. 从有条形码的一端撬开三明治结构。 Sandwich structure from one end pry bar code. 通过将干净镊子伸入两张拨片之间,轻轻分开两张玻片。 By extending into a clean forceps between the two paddles, gently separate the two slides. 让垫片落入染色缸底部。 Let the gasket falls into the bottom of the staining jar. 将芯片移至染色缸2#(Wash B uffer I)的芯片架上。 The chips move staining jar 2 # (Wash B uffer I) of the chip rack. 同时避免让芯片暴露于空气中。 Let chip while avoiding exposure to air.

[0056] 开启磁力搅拌器搅拌5min。 [0056] Open magnetic stirrer 5min. 调整转速以达到良好而不过分的搅拌。 Adjusting the speed to achieve a good and not too much agitation. 将芯片架转移至装有Wash Buffer 2、预热到37°C的染色缸3#,搅拌lmin。 The carrier was transferred to a chip with Wash Buffer 2, staining jar preheated to 37 ° C # 3, stirred for lmin. 在5_10s内缓慢将芯片架移出。 The chip holder was slowly removed in 5_10s. 尽可能减少芯片上的液滴。 Minimize droplet on the chip. 弃用过的Wash Buffer I和Wash Buffer 2。 Banned in the Wash Buffer I and Wash Buffer 2. 立即扫描芯片以减少环境中氧化剂对于信号强度的影响。 Now the chip scan the environment to reduce the effect of the oxidizing agent to the signal strength. 本实施例的基因芯片操作步骤简单,检测特异性高,稳定性好,该芯片多次试验重复性高;时间短,从样本抽提到获得扫描结果可在一天内完成。 GeneChip Operating procedure of this embodiment is simple, high detection specificity, stability, repeatability test multiple chip; short time, from a sample can be extracted into the scan results obtained in one day.

Claims (5)

  1. 1.一种检测Angelman综合征的基因芯片,包括固相载体和固定在该固相载体上的寡核苷酸探针,其特征在于,所述寡核苷酸探针为SEQ ID N0.1~50所示的核苷酸序列。 1. A method of detecting Angelman syndrome gene chips, oligonucleotide probe comprising a solid support and immobilized on the solid support, wherein said oligonucleotide probe is SEQ ID N0.1 ~ 50 nucleotide sequence.
  2. 2.根据权利要求1所述的检测Angelman综合征的的基因芯片,其特征在于,所述固相载体选自载玻片、硅片、硝酸纤维素膜、尼龙膜或聚苯乙烯膜。 The gene detecting chip according to claim Angelman syndrome, wherein said solid support is selected from glass slides, silicon, nitrocellulose membrane, nylon membrane or a polystyrene film.
  3. 3.一种用于检测Angelman综合征的试剂盒,其特征在于,所述试剂盒包括权利要求1所述的基因芯片。 3. A kit for detecting Angelman syndrome, wherein said kit comprises a gene chip according to claim 1.
  4. 4.根据权利要求3所述的用于检测Angelman综合征的试剂盒,其特征在于,所述试剂盒还包括标记物,所述标记物为Cy3-dUTP。 According to claim kit for detecting Angelman syndrome claim 3, wherein said kit further comprises a label, the label is Cy3-dUTP.
  5. 5.根据权利要求3所述的用于检测Angelman综合征的试剂盒,其特征在于,该试剂盒还包括标记物,所述标记物为Cy5_`dUTP。 5. A kit for detecting claim Angelman syndrome claim 3, wherein the kit further comprises a marker, the marker is Cy5_`dUTP.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085752A1 (en) 2000-04-29 2001-11-15 Shanghai Biowindow Gene Development Inc. A novel peptide-human myosin heavy chain 12-14 and the polynucleotide coding this novel peptide
WO2005111237A1 (en) 2004-05-05 2005-11-24 Biocept, Inc. Detection of chromosomal disorders

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085752A1 (en) 2000-04-29 2001-11-15 Shanghai Biowindow Gene Development Inc. A novel peptide-human myosin heavy chain 12-14 and the polynucleotide coding this novel peptide
WO2005111237A1 (en) 2004-05-05 2005-11-24 Biocept, Inc. Detection of chromosomal disorders

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