Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.
Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.
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DOI：
10.1038/bjc.1993.236
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发表时间：
1993-06
影响因子：
8.8
通讯作者：
Quirke, P
中科院分区：
文献类型：
--
作者：
Cawkwell, L;Bell, S M;Lewis, F A;Dixon, M F;Taylor, G R;Quirke, P
为了研究结直肠肿瘤中的等位基因丢失，我们开发了一种快速技术，该技术克服了与等位基因丢失的放射性限制性片段长度多态性（RFLP）分析相关的大多数问题。我们利用微卫星长度多态性，这是高度信息化，并密切相关的目的基因座。包含微卫星的序列可以通过聚合酶链反应（PCR）从正常和肿瘤DNA对中扩增出来，其中一个引物被荧光标记。这使我们能够使用专用软件在自动DNA测序仪上运行的聚丙烯酰胺凝胶上检测产物，通过该软件，结果根据峰大小，高度和面积自动定量。使用这种技术，我们已经分析了26个正常组织：癌症对等位基因损失在两个位点的腺瘤性息肉病结肠（APC）基因染色体5q上。重复测定对每对产生了相同的结果。在25个有信息的样本中，有10个（40%）发现等位基因丢失。
In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).