High-throughput analysis of fragile X (CGG)(n) alleles in the normal and premutation range by PCR amplification and automated capillary electrophoresis
High-throughput analysis of fragile X (CGG)(n) alleles in the normal and premutation range by PCR amplification and automated capillary electrophoresis
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DOI：
10.1007/s004390050552
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发表时间：
1997-10-01
期刊：
影响因子：
5.3
通讯作者：
Vuust, J
中科院分区：
文献类型：
--
作者：
Larsen, LA;Gronskov, K;Vuust, J
脆性X综合征是由FMR1基因转录本5‘非翻译区内的(CGG)(N)三核苷酸重复序列扩大引起的。Southern blotting可以可靠地诊断该病，但这种方法工作量很大，需要大量样本。因此，对于大多数样本正常的大型研究或筛选项目，需要一种更快速、更便宜的方法。我们提出了一种在正常和前突变范围内对FRAXA(CGG)(N)区域进行准确、高通量分析的方法。该方法是基于从全血中提取的DNA或从滤纸上的干血斑中洗脱的DNA进行聚合酶链式反应(PCR)扩增，然后自动毛细管电泳法和多色荧光检测。该方法允许在48小时内处理144个样品，分析内测定的准确度为0.2-1.8个碱基对。我们对30名患者的样本进行了盲法再分析，这些样本以前是通过Southern blotting或带有放射性标记的PCR进行分析的。在这项研究中，根据(CGG)(N)重复次数正确地确定了正常和前突变等位基因，范围从28-121(CGG)(N)重复。杂合子样本中的所有全突变等位基因和一个大的前突变等位基因都未能扩增。该方法被用来确定FRAXA(CGG)(N)重复序列在丹麦人群中的分布。
Fragile X syndrome is caused by expansion of a (CGG)(n) trinucleotide repeat within the 5' untranslated region of the FMR1 gene transcript. The disease is reliably diagnosed by Southern blotting, but this method constitutes a significant workload and requires large samples. Therefore, for large research or screening projects in which a large majority of the samples will be normal, a more rapid and less expensive method is needed. We present a method for accurate, high-throughput analysis of the FRAXA (CGG)(n) region in the normal and premutation range. The method is based on polymerase chain reaction (PCR) amplification of DNA extracted from whole blood or eluted from dried blood spots on filter-paper followed by automated capillary electrophoresis and detection by multicolour fluorescence. This method allows a throughput of 144 samples in 48 h, with an intra-assay accuracy in size determination of 0.2-1.8 bp. We performed a blind reanalysis of samples from 30 patients, previously analysed by Southern blotting or PCR with radioactive labelling. In this study normal and premutation alleles, ranging from 28-121 (CGG)(n) repeats, were correctly determined with respect to number of (CGG)(n) repeats. All full-mutation alleles and one large premutation allele in a sample of a heterozygote failed to amplify. The method was used to determine the distribution of FRAXA (CGG)(n) repeats in the Danish population.