Name: simple method for fluorescence dna in situ hybridization to squashed chromosomes
Uploaded: 2015-01-06T10:15:00.000+00:00
Duration: 11 min 36 s
Description: Watch this Scientific Journal Video about Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes at JoVE.com

We thank Zhaohua Irene Tang in the W. M. Keck Science Department for the use of her epifluorescence microscope and the Werren lab for donating Nasonia for dissections. This work was supported in part by an NIH-NRSA fellowship (5F32GM105317-02) to AML.

1.组织解剖和固定（60分钟） <ol><li>对果蝇的大脑，放在第三龄幼虫在下降的1×PBS（磷酸盐缓冲盐水）中。选择大的3 龄幼虫正在积极从小瓶或瓶子是不是人满为患爬行。 <ol><li>用一个镊子超细对抓牢口钩，另一镊子对抢2/3下来身体（ 图1A，B）的长度。上嘴钩轻轻一拉，露出大脑，腹侧神经节，唾液腺及部分幼虫消化道。使用镊子分离脑和腹侧神经节（ 图1A，B）中从其他组织中，并装在1倍的PBT（磷酸盐缓冲盐水与吐温）上的塑料培养皿的液滴。 </li></ol></li><li>对于金小蜂睾丸的解剖，选择男3日龄蛹（黄体红色的眼睛）。男金小蜂有小翼片的长度相对到f在蛹期（ 图1C）emales。 <ol><li>握住蛹在与一个镊子对，并使用其他镊子对胸部区域附近的腹部的上，抓住腹部的很远尖端和拉出泪滴形睾丸（它们将被脂肪包围体，可以轻轻摇动走; 图1D）。断开任何外部车身零件的睾丸，这将妨碍正常的挤压，并将其放置在1X PBT上的塑料培养皿液滴。 </li></ol></li><li>对于每一个幻灯片，解剖一共有四五个组织样本（ 即 ， 果蝇幼虫大脑和睾丸金小蜂 ）。 注:以上五个样本会导致过度拥挤的细胞核和染色体。 </li><li>可选地，为了实现在有丝分裂的染色体常染色质区域姐妹染色单体的一些分离，治疗用低渗溶液的组织:从1倍的PBT（传送到大脑的下降为0.5％的柠檬酸钠5-10不超过10）分钟。 注:秋水仙碱（有丝分裂抑制剂）可以增加核分裂象数量在中期11有益的。但是，它的使用是不必要的，如果非常大的健康的幼虫被使用，并且甚至不希望的，因为它可以在染色体分辨率产生负面影响，传播和形态。 </li><li>放置的固定液（45％乙酸的2.5％多聚甲醛）倒在干净Sigmacote处理的盖玻片的表面上的液滴（约20微升）。 注意:请固定液新鲜使用的每一天。固色剂解决方案，从1.8-3.7％多聚甲醛在45％的醋酸产量FISH最好的结果。用面巾纸比大脑和睾丸等，或者此适配协议免疫FISH可能需要与不同的固定剂试验（针对不同的固定剂的列表，请参阅12）。 </li><li>小心地从解剖缓冲液（1×PBT）每一组织样品与超细镊子，米尼米转移到固定剂液滴查懋解剖缓冲器入固定液的转移。定位的组织样本，使得它们被均匀地彼此间隔开的固定剂液滴内。孵育在固定液组织在室温下4分钟。 </li><li>小心地将聚赖氨酸涂覆的滑动面向下到组织和盖玻片。不按下在这一点上，而是允许两个接触轻轻使盖玻片粘到滑动的下侧。倒置滑动，使得盖片是在顶部。 </li><li>夹心折叠一张滤纸内部幻灯片/组织/盖玻片。在一个稳定的表面，用拇指，按非常坚定地直下到盖玻片上方的位置。小心避免横向盖玻片滑动（这将导致涂抹组织）。 </li><li>淹没在滑动/组织/盖玻片入液氮（ 见图1E，女装置），并且静置直至氮停止沸腾（长为fINE）。取出载玻片，并立即通过轻弹盖玻片的角在向上方向上（避免刮伤固定的组织与剃刀刀片）折断盖玻片用新鲜刀片。 <ol><li>预冷幻灯片/组织/盖干冰块上滑，盖滑起来，1-2分钟浸入液态氮将有助于防止开裂的幻灯片之前。 </li></ol></li><li>立即用纸巾滑动放入一个科普林缸填充100％的乙醇在室温下，静置至少5分钟（如果需要，这个时间可以更长）。 注:冷100％乙醇也可以使用。 </li><li>删除与组织滑动，毛细作用带走用Kimwipe过量乙醇（而不触及固定的组织），并离开该滑动风干1小时。 <ol><li>直接进入第2步或保存幻灯片干燥低湿度空气中或在干燥室周来进行杂交之前甚至几个月。 </li></ol></li></ol> 图1:与在那里抢口钩显示位置（A）A 3 路龄幼虫果蝇 （右一），（表示带*号）和下幼虫解剖大脑的方式2/3; （左）的概略幼虫一个相同发育阶段的，描绘了幼虫头部内脑的相对位置。（B）中的脑和腹侧神经节从一个第三龄果蝇幼虫（右）和本示意解剖组织（左）。（C）3日龄蛹金小蜂在黄体红眼阶段。（D）睾丸对从3日龄金小蜂的蛹男（右）与解剖位置，表示从哪里抢蛹在腹部的后（标以*）和中途上的主体; （左）示意描绘男性和女性是P蛹;雄性蛹可以通过翼不延伸超过矢状轮廓（黑色箭头），而相比之下，雌性，其具有翼延伸过去的信息来区分;睾丸对的相对位置示于阳蛹。纸夹-和（F）的方法，用于浸泡在液氮的容器幻灯片（E）的装置， -一个字符串。可用于对多张幻灯片同时浸没多个回形针字符串。 2. 在原位杂交（30月1日分; 1小时，2.5小时长探头，第2天） <ol><li>加入1μl每种探针的（100毫微克），以20微升1.1倍的杂交缓冲液中。吸管探针/杂交缓冲液到固定组织的表面（避免触及组织）。 </li><li>小心直接置于盖玻片上的探针/杂交缓冲液中，并确保盖玻片工作在组织直接居中。该缓冲区应该迁移到THË盖玻片的外缘，不留气泡。 注:小气泡不接触组织不会造成任何问题的程序。通过仔细抬起盖玻片的一个角落里，小心地放弃它返回到幻灯片中删除大量气泡。 </li><li>将幻灯片/组织/盖玻片到块预加热到95°C（盖滑起来）的表面。有一大块铝箔盖，以防止曝光。让滑动孵育在95℃下5分钟。 注意:有孔典型热块管可以翻转，提供在其上放置幻灯片/组织/盖玻片一个平面上。 </li><li>去除幻灯片，让它稍微冷却，直到它是温暖的触感。精心包装了一块拉伸封口膜周围的盖玻片密封它下面的液体。 </li><li>放置一个湿度室内部的密封滑动，并把该室到预热至30℃的培养箱。孵育在30℃下进行4小时至过夜。<ol><li>创建湿度室从空尖框或带盖百惠容器放置在底部阻尼的Kimwipes或纸巾。 注:单链DNA寡核苷酸探针被设计为28-33个碱基，达到45-47℃的理论上的熔解温度（T M）。这些长度和T 米范围反映的事实是，我们已经研究了很多重复序列是富含AT的，因此具有非常低的GC含量。较长的探针将可能有较高的Tm 值值;这可能会导致在30℃的标准杂交温度更高的背景杂交。因此，随着杂交温度一些故障，可能需要以达到最佳的效果。找到最好的杂交温度，增加（或减少）由5℃的温度下，增量。 </li></ol></li><li>仔细从幻灯片中删除封口膜，然后慢慢抬起一个角落里小心地取出盖玻片。华SH幻灯片三次15分钟的0.1X SSC缓冲每次洗。在洗涤覆盖科普林缸用铝箔，以减少曝光。 </li><li>如果不使用长生物素化的探针，继续执行步骤2.8。 <ol><li>如果使用的是长的生物素化的探针，干周围用Kimwipe组织的区域，小心不要触碰组织本身。放置100微升封闭溶液在组织中并用盖玻片覆盖轻轻，注意避免俘获气泡。包住滑过盖玻片用Parafilm和地点，在37℃下进行30分钟。 </li><li>小心地取出盖玻片和周围涂抹用Kimwipe组织。吸取100μl的若丹明​​ - 亲和素稀释1:1000在SBT过组织并覆盖轻轻用盖玻片，注意避免俘获气泡。包住滑过盖玻片用Parafilm和地点，在37℃下进行30分钟。 </li><li>小心地取出盖玻片和5分钟洗滑3次，每次在4倍SSCT然后3次，每次5分钟，在0.1×SSC。 注:幻灯片可水洗的时间较长。 </li></ol></li><li>去除幻灯片及吸干组织周围用干的Kimwipe以去除多余的缓冲液（避免接触的组织）。放置在滑动组织朝上，在暗处10-15分钟或直至水分完全消散。 </li><li>吸管11微升的Vectashield封固剂（有4&#39;，6-二脒基-2-苯基 - DAPI）到组织上。小心地将一个干净的盖玻片（不与Sigmacote处理）的正上方的安装介质和组织的核心。安装介质应当向外迁移缓慢地向盖玻片的边缘。 注:如果在安装介质无法到达所有各方盖玻片的边缘，再追加1-2微升封固剂可以施加到一个位置，在该盖片的边缘，以填补在所需体积。在这种情况下，可以肯定之前擦去从滑动表面上的任何多余的介质密封。 </li><li>密封盖玻片指甲油的边缘。避免涂抹指甲油在组织样本。 </li><li>放置在滑动直立在暗处，并让指甲油干燥直至完全硬（通常为30分钟以上）。在这一点上，图像中的组织或储存在-20℃下长达1周后成像。 </li></ol>缓冲/解决方案食谱 10X PBS <ul><li> 80克氯化钠</li><li> 2.0克氯化钾</li><li> 14.4克的Na 2 HPO 4 </li><li> 2.4克KH 2 PO 4的</li><li> pH值至7.4，H 2 O到1升</li></ul> 1X PBT <ul><li> 5毫升10X PBS </li><li> 45毫升H 2Ø </li><li> 0.1％吐温20 </li></ul> 20X SSC <ul><li> 175.3克氯化钠</li><li> 88.2克柠檬酸钠</li><li> 800毫升水2 O </li><li> pH值7，H 2 O到1升</li></ul> 4X SSCT <ul><li> 200毫升20X SSC </li><li> 799毫升的H 2 </子&gt; 0 </li><li> 0.1％吐温20 </li></ul> 0.1X SSC <ul><li> 5毫升20X SSC </li><li> 995毫升H 2Ø </li></ul>杂交混合物（20微升;从11修改） <ul><li> 10微升甲酰胺</li><li> 4微升50％硫酸葡聚糖</li><li> 2微升20X SSC </li><li> 4微升水中2 O </li></ul> SBT 8（10毫升）中<ul><li> 2毫升20X SSC </li><li> 0.01克牛血清白蛋白（BSA）的</li><li> 10微升吐温20 </li><li> 7.9毫升H 2Ø </li></ul>封闭溶液8（10毫升）中<ul><li> 0.3克BSA </li><li> 10微升吐温20 </li><li> 2毫升20X SSC </li><li> 8毫升H 2Ø </li></ul>用多聚甲醛固定液（1毫升）中<ul><li> 393.75微升H 2 O（先加水） </li><li> 450微升冰醋酸</li><li> 156.25微升16％多聚甲醛</li></ul>

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The authors declare that they have no competing financial or any other conflict of interest.

DNA的原位杂交是经常用于映射特定序列的染色体。我们描述了一个简单的方法DNA ISH高拷贝数，异色序列优化。而不是使用洗涤的甲酰胺溶液中，这是在其他现有的DNA ISH协议的要求，我们直接放置在预加热块组织安装滑动到使DNA变性。这种方法绕过了使用大量的甲酰胺。用于产生脆的杂交信号的一个关键步骤是使用新鲜制备的固定溶液;如果不这样做（或者使用多聚甲醛是已经开放超过一个月新溶液）可能会降低信号的分辨率。这种方法的一个限制是，像用于其它DNA ISH协议中，杂交温度，可能需要优化，以消除杂散杂交脱靶序列。 这个协议是结合有单fluorop敏感短路，单链DNA探针每分子hore可以检测重复的块与甚至相对低的重复拷贝数（ 例如，〜700 Rsp时重复; 图2C）。信号，以具有较少的重复卫星块可被可视化的短单链DNA探针通过使用一个高度敏感的数码相机。虽然这里未示出，这种方法也是有效的在常染色质10映射的非重复序列。使用长，生物素化的探针提供了在检测到低拷贝数序列作为信号可以容易地通过重复处理来扩增更灵活的生物素化的抗生物素蛋白和罗丹明-抗生物素蛋白9。为了显现单拷贝序列，多个探针跨越序列可能是必要的。或者，含有目的序列的细菌人工染色体（BAC），可以通过切口平移或随机引物用作探针用于非重复序列10进行标记。要映射序列在判罚尺度，探头瞄准differen吨重复（每个具有不同的荧光团）可同时使用，以获得相对于其它重复和在染色体的结构标靶序列的位置。而我们使用两个探针此处（ 图2A，B，D），探针的数目可以容易地提高到3 10个或更多，这取决于可用的不同显微镜的荧光过滤器的数量。该协议可以扩展到其他组织，我们已经验证了协议的工作以及在多线染色体南瓜从唾液腺。可以想象的是，这种方法可以结合免疫染色的染色质蛋白;然而，建议免疫染色来执行作为第一步骤，并随后重新固定用多聚甲醛的DNA ISH之前，以防止DNA的原位杂交中的抗体损失。

原位杂交（ISH的DNA）的DNA是一种常用的方法进行映射的序列，以特定的染色体区域。可以通过少数的方法，包括缺口转译或长DNA产物1,2-末端标记和deoxygenin（DIG）的掺入-attached核苷酸及其识别通过各种各样的产生内常染色质探针，以单拷贝区基的缀合抗体1-3。在几个或单拷贝数常染色体序列的可视化需要使用任何单一的大型探测器具有高比活或多个较小的探针共同增强信号的鸡尾酒。 与此相反，在异发现高度重复序列，诸如卫星的DNA，是用于DNA ISH更容易的目标，因为它们通常作为几十到重复聚集在被称为块的单染色体区域的数千存在。转座元件也可以是在不同的时染色体位点2个高拷贝数发现。在这些情况下，单个探针具有低比活性可以有效标记异色序列由于在多个位点的杂交。探针的重复序列可被合成市售的短的寡核苷酸（30-50碱基对）和化学缀合与任何多种不同的荧光团。通过基因组测序技术绘制内异染色质重复序列是困难的，因为在高度重复的卫星模块4-6,7建筑脚手架遇到的挑战。目前，ISH站作为在子染色体水平映射这些序列的最有效的方法。这种策略是映射大批正在通过发现正在进行的基因组和转录组测序研究的重复序列的重要。 映射重复序列上滑动安装染色体的效率和易于将选取gre通过对DNA ISH简化协议atly增强。例如，对于DNA的ISH现有的协议涉及杂交的组织中的甲酰胺溶液2,8-多次洗涤，从而增加大致映射序列所需的时间，也产生这种昂贵试剂的大量化学废物。在这里，我们描述了一个修订后的DNA ISH方法规避，需要甲酰胺清洗，并利用基本的实验室设备和试剂。该方法最初是通过使用缀合有荧光染料商业合成的寡核苷酸设计为在果蝇幼虫的神经母细胞异染色质区域高度重复的DNA序列的快速映射。但是，这种方法也适用于通过使用通过其他方式9,10和跨多个不同的组织和染色体类型合成更大的探针映射重复序列。此外，此方法可用于通过使用较长或MULTIP映射常染色质的序列乐，感兴趣的常染色体序列中短探针。

<table><tbody><tr><td>Poly-L-lysine coated slides (regular slides also can be used)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Ultrafine tweezers (5 gauge)</td><td>Dumont</td><td></td><td></td></tr><tr><td>22 x 22 mm cover slips</td><td>Fisher</td><td></td><td>Sigmacote-treated by immersion for 15 sec, blotting dry, and wiping away all traces of Sigmacote so that cover slip is clear</td></tr><tr><td>Sigmacote</td><td>Sigma</td><td></td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td>75 - 150 mm</td></tr><tr><td>Paraffin wax paper</td><td></td><td></td><td></td></tr><tr><td>Heat block with thermometer</td><td></td><td></td><td></td></tr><tr><td>Dry incubator</td><td></td><td></td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Humidity chamber</td><td></td><td></td><td>empty pipette tip box or Tupperware, lined with moistened paper towels or Kimwipes</td></tr><tr><td>Coplin jars</td><td></td><td></td><td>with slide grooves</td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml microfuge tubes</td><td></td><td></td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td>clear or colored</td></tr><tr><td>P20 micropipette and plastic tips</td><td></td><td></td><td></td></tr><tr><td>Paperclips</td><td></td><td></td><td>20 - 25 standard metal paperclips linked to form a chain</td></tr><tr><td colspan="4">Reagents</td></tr><tr><td>16% EM grade paraformaldehyde</td><td>Electron Microscopy Reagents</td><td></td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>100% Ethanol, chemical grade</td><td></td><td></td><td></td></tr><tr><td colspan="4">Commercially synthesized, fluorescently labeled oligos</td></tr><tr><td>Long biotinylated probe</td><td>Invitrogen; Alternative steps 2.7.1-2.7.3</td><td></td><td>&lt;em&gt;e.g.,&lt;/em&gt; nick translated and biotinylated with BioNick from Invitrogen</td></tr><tr><td>Rhodamine-Avidin</td><td>Roche; Alternative steps 2.7.1-2.7.3</td><td></td><td>for detection of long biotinylated probe</td></tr><tr><td>Hybridization buffer</td><td>Recipe above</td><td></td><td></td></tr><tr><td>4x SSCT</td><td>Recipe above</td><td></td><td>saline-sodium citrate + Tween</td></tr><tr><td>0.1x SSC</td><td>Recipe above</td><td></td><td>saline-sodium citrate</td></tr><tr><td>Blocking solution</td><td>Recipe above</td><td></td><td></td></tr><tr><td>SBT</td><td>Recipe above</td><td></td><td>SSC, bovine serum albumin, Tween</td></tr><tr><td>1x PBT</td><td>Recipe above</td><td></td><td>phosphate-buffered saline + Tween</td></tr><tr><td>1x PBS</td><td></td><td></td><td>phosphate-buffered saline</td></tr><tr><td>Hypotonic solution</td><td></td><td></td><td>0.5% sodium citrate in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Formamide</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector laboratories</td><td></td><td></td></tr></tbody></table>

simple method for fluorescence dna in situ hybridization to squashed chromosomes

原位杂交（ISH的DNA）的DNA是一种常用的方法进行映射的序列，以特定的染色体区域。这种方法是在映射高度重复序列，以异染色质区域，其中，计算方法面临令人望而却步的挑战特别有效。在这里，我们描述了一个精简的协议，用于DNA ISH是规避洗甲酰胺是在其他DNA ISH协议的标准步骤。我们的协议被优化用于与携带荧光染料，从而有效地标记在多个不同的昆虫组织类型的异色染色体区域内的重复DNA序列的短单链DNA探针杂交。然而，应用程序可被扩展较大探针和单拷贝（非重复）的DNA序列的可视化中使用。我们证明了该方法通过映射几个不同的重复序列从果蝇染色体挤压神经果蝇细胞和金小蜂vitripennis精母细胞。我们表明杂交模式均小，商业合成的探针和用于比较的较大探针。此过程使用简单的实验室耗材和试剂，是理想的谁与执行DNA ISH一点经验调查。

为了证明这种方法，我们杂交的一组小，以致被化学修饰的荧光偶联物（ 图2）和一个较长的生物素化的探针商业合成的寡核苷酸（通过PCR产物的切口平移制成; 图2B），以染色体从几个不同的组织类型（ 见表1）。目标序列包括位于从D.有丝分裂染色体着丝粒（异）地区的卫星重复果蝇幼虫神经母细胞（ 图2A-C）和减数分裂染色体从蛹N. vitripennis睾丸（ 图2D）。在每一种情况下的杂交揭示离散的带型在子的染色体区域。值得一提的是，探头的D.果蝇 359 bp的卫星重复识别在X既有大型多兆碱基对的块，并在3号染色体小得多区域（ 图2A）。对响应卫星更长的探头还检测到重复的块组成的相对较低的重复拷贝数（〜700份; 图2B）。因此，该方法允许非常小的卫星块可视化。为了帮助揭示染色体结构，并有助于染色体鉴定，它可以是治疗染色体用低渗溶液（在步骤1.5中所述）是有用的。用低渗溶液，在图板2B和2C的组织进行处理5分钟和10分钟分别。注意姐妹染色单体的分离是更大的更长的治疗（ 图2C）。 <img alt="图2" src="/files/ftp_upload/52288/52288fig2highres.jpg" width="600" /> 图2:DNA 原位杂交（A）从D.幼虫成神经细胞的染色体果蝇 /D. simulans杂种;（B）和（C）的幼虫成神经细胞从D染色体果蝇治疗低渗溶液（见解剖和固定步骤1）;（D）从宝石黄蜂金小蜂vitripennis蛹睾丸组织中减数分裂染色体。在（A）中 ，绿色箭头表示AATAT卫星上的D.小区域simulans 第 4染色体，而绿色箭头指向AATAT上D.果蝇 X染色体。红色箭头突出的359 bp的卫星小区域的D.果蝇 第三染色体，而红色箭头标记359 bp的序列的十中（A）的所有卫星进行杂交短，荧光标记的寡核苷酸。在（B）中 ，在绿信号对应于AAGAG卫星（合成低聚）和红色信号是120bp的响应卫星（生物素化的探针;标用红色箭头）。（C）的同样的响应卫星具有短荧光标记的寡（红色箭头）。在（D），红色箭头标志着卫星是唯一位于父系性别比（PSR）染色体，这是N的自然种群中的非必需的，编外B染色体vitripennis 13，而绿色箭头表示对正常的染色体中的一个卫星;这种杂交通过使用短，荧光标记的寡核苷酸进行。所有探头都详见表1。比例尺面板（A）为10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52288/52288fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 探测器名称 </td><td> 探头类型 </td><td> 寡 </td><td><stroNG&gt;标签</td></tr><tr><td> AAGAG卫星</td><td>寡</td><td> （AAGAG）7 </td><td> 5&#39;6-FAM（荧光素） </td></tr><tr><td> AATAT卫星</td><td>寡</td><td> （AATAT）6 </td><td> 5&#39;-Cy3的</td></tr><tr><td> 359-BP卫星</td><td>寡</td><td> TTT TCC AAA TTT CGG TCA TCA AAT AAT CAT </td><td> 5&#39;-的Alexa555 </td></tr><tr><td> PSR卫星</td><td>寡</td><td> TGT AAC TGG AGG AAA AAA ATG TAT TAT TGA </td><td> 5&#39;-的Alexa555 </td></tr><tr><td>金小蜂常染色体卫星</td><td>寡</td><td> AAT TTT GTG AAT TTT GGT GTC TCC ATC </td><td> 5&#39;-Alexa633 </td></tr><tr><td rowspan="2">响应</td><td rowspan="2"> PCR </td><td> F-5&#39;AAA GGA TCA CCC ATT TTG ATC GC </td><td rowspan="2">生物素 - 14的dATP </td></tr><tr><td> R-5&#39;CCG AAT TCA AGT ACC AGAÇ </td></tr></tbody></table>表1:探针在图2中使用的类型。

Watch this Scientific Journal Video about Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes at JoVE.com

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

simple-method-for-fluorescence-dna-situ-hybridization-to-squashed

Universidad San Sebastian

Research

JoVE Journal

Biology

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

18.0K 次观看.  University of Rochester. Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

视频: Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

科研

遗传学

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

3D多色DNAFISH工具研究人类原细胞核结构

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

生物学

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

果蝇聚乙烯南瓜抗体标记染色体的制备

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

DNA晕片制备物的荧光原位杂交揭示整个染色体、端粒和基因位点

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells

Source: Wei Liu&#160;et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58950"> Merkel Cell Polyomavirus Infection and Detection</a>, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions were subjected to in situ hybridization chain reaction to detect MCPyV specific genomes. Further, the DNA-HCR technique was combined with FISH to visualize the MCPyV DNA in infected human skin cells.

Source: Wei Liu&#160;et al.,  Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions ...

对于荧光DNA的简易方法

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