快速风干滴染色体制备方法适用于FISH在植物
Summary
A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.
Abstract
染色体利差的准备是荧光原位杂交技术的成功表现（FISH）的先决条件。高品质的植物染色体利差准备是具有挑战性，因为刚性细胞壁。之一的植物染色体的制备的批准的方法是所谓的滴制剂，也称为滴扩展或空气干燥技术。在这里，我们提出了一个协议，为快速编制有丝分裂染色体的利差适合FISH检测单和高仿DNA探针。这种方法是在50％-55％的相对湿度下进行的风干滴法的改进的变体。这个协议包括清洗步骤使得其应用简单，有效和可再现的数目减少。这种方法的显而易见的好处是作为成功的FISH分析了完善的前提条件良好的蔓延，没有损坏和大量的中期染色体。使用这个协议，我们得到高品质的CHROMosome差价和可重复的FISH结果大麦，H球茎，H海鱼，H murinum，H pubiflorum和黑麦 。
Introduction
荧光原位杂交（FISH）是一种有效的工具，单一的，高拷贝序列在染色体水平的物理图谱。前提条件是高品质的染色体利差的准备。没有一般染色体制备方案，这将是同样适用于动物和植物细胞。植物染色体的制备是特别具有挑战性，因为不同的物种中的刚性细胞壁和各种细胞质的一致性。之一的有利方法植物染色体的制备是一个所谓的滴技术也被称为落扩频技术和空气干燥技术1,2。该方法首先在1958引入由Rothfels和Siminovitch用于体外生长哺乳动物细胞3。后来Martin等人4和Kato等人5适于这种方法的植物。
最近，一个名叫法“SteamDrop＆＃39;被开发了用于水蒸汽的非重叠的染色体6的制备。虽然，前面7观察到高湿度的积极影响，'SteamDrop'提供高质量染色体制剂6的受控流。蒸汽处理引起拉伸可能连接到染色体蛋白质的一些修改染色体。导致中期扩展的质量是非常高的，虽然中期完成足够数量的固定利差随后FISH实验要求的专业技术。
在这里，我们提出了一个协议为适合FISH检测单和高仿探测器5,8的有丝分裂染色体谷物的准备。这种方法是由加藤下的50％-55％的相对湿度进行9（图1）中描述的风干落下法的改进的变体。这个协议包括减少数量的洗涤步骤使得其应用简便，高效和可重复性。使用这个协议，我们得到高品质的染色体利差和FISH结果大麦，H球茎，H海鱼，H murinum，H pubiflorum和黑麦 。
Protocol
Representative Results
Discussion
染色体制备实验已进行了使用属于禾本科（禾本科）谷物幼根。所有分析的品种有14相对较长的有丝分裂中期染色体（11-15微米）的二倍体基因组集，属于大型基因组的物种（5.1-7.9 GBP）的。
发芽根长度不超过2厘米，以获得最大的分生组织。分裂细胞同步是由一个20小时长冰-水处理该改进的有丝分裂中期的扩散量10来实现的。
两个步骤是在制?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.
Materials
| Hot Plate | MEDAX GmbH | 12603 | |
| Cellulase R10 | Duchefa | C8001 | |
| Cellulase | CalBioChem | 219466 | |
| Pectolyase | Sigma | P3026 | |
| Cytohelicase | Sigma | C8274 | |
| Texas Red-12-dUTP | Invitrogen | C3176 | direct fluorochrome |
| Fluor488-5-dUTP | Invitrogen | C11397 | direct fluorochrome |
| Fluorecsence microscope | Olympus BX61 | BX61 | |
| CCD camera | Orca ER, Hamamatsu | C10600 | |
| 4’,6-diamidino-2-phenylindole (DAPI) | Vector Laboratories | H-1200 | fluorecsent dye |
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