A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants
A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants
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DOI：
10.3791/53470
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发表时间：
2015-12-01
影响因子：
1.2
通讯作者：
Houben, Andreas
中科院分区：
文献类型：
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作者：
Aliyeva-Schnorr, Lala;Ma, Lu;Houben, Andreas
染色体扩增片的制备是荧光原位杂交（FISH）成功进行的先决条件。由于细胞壁坚硬，制备高质量的植物染色体扩增具有挑战性。经批准的植物染色体制备方法之一是所谓的滴法制备，也称为滴法扩散或风干技术。在这里，我们提出了一种快速制备有丝分裂染色体扩散的方案，适用于单拷贝和高拷贝DNA探针的FISH检测。该方法是在相对湿度为50%-55%的条件下进行的空气干滴法的改进变体。该协议包括减少数量的洗涤步骤，使其应用简单，高效和可重复。这种方法的明显好处是扩散良好，未损坏和大量的中期染色体是成功进行FISH分析的完美先决条件。使用该方案，我们获得了高质量的染色体扩增和可重复的FISH结果，包括普通Hordeum vulgare， H. bulbosum， H. marinum， H. murinum， H. pubbiflorum和seale cereale。
Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.