Method for the purification of tissue DNA suitable for PCR after fixation with Bouin's fluid - Uses and limitations in microsatellite typing
Method for the purification of tissue DNA suitable for PCR after fixation with Bouin's fluid - Uses and limitations in microsatellite typing
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DOI：
10.1097/00019606-199706000-00007
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发表时间：
1997-06-01
影响因子：
--
通讯作者：
Moynet, D
中科院分区：
文献类型：
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作者：
Longy, M;Duboue, B;Moynet, D
石蜡包埋组织通常是在各种医疗目的中执行基于聚合酶链式反应(PCR)分析的唯一可用的材料。不幸的是，在许多国家，酸固定剂的使用限制了这种材料在分子分析中的使用。本文报道了一种从Bouin固定和石蜡包埋的样品中提取DNA的方法学细节，该技术基于DNA的双重洗涤，在酒精中，然后在水介质中，这使得从该材料进行PCR反应成为可能。与有机溶剂纯化冰冻组织DNA的结果相比较，在琼脂糖凝胶上检测扩增产物方面显示出良好的重复性。然而，这两种方法之间的差异在微卫星序列(CA重复)的等位基因分型谱中非常常见，要么是作为新的等位基因，要么是由于固定材料中正常等位基因的丢失，这构成了使用Bouin固定组织的DNA作为精细等位基因分型的底物的限制。
Paraffin-embedded tissues are often the only available material to perform polymerase chain reaction (PCR)-based analysis in various medical purposes. Unfortunately, the use in many countries of acid fixatives such as Bouin's fluid limits the use of such a material for molecular analysis. This article reports the methodological details of a DNA purification technique from Bouin-fixed and paraffin-embedded samples based on a double washing, in an alcohol then in an aqueous medium, of the DNA, which enables PCR reactions from this material. Comparison of the results with those obtained by organic solvent purification of DNA from frozen tissue fragments showed excellent reproducibility in terms of detection of an amplification product on agarose gel. However, differences between the methods were quite frequently seen in the allelic typing profile of microsatellite sequences (CA repeats), either as neo-alleles or by the loss of normal alleles in the fixed materials that constitute a limitation in using DNA from Bouin-fixed tissue as a substrate for fine allelotyping.